Vascular access preservation in hemodialysis patients

ABSTRACT

The present invention relates generally to the field of hemodialysis, including methods and kits that can be employed to improve hemodialysis therapy. The present invention encompasses methods and kits useful for reducing vascular access complications associated with hemodialysis therapy and prolonging the period of time for which a vascular access site can be used in a patient.

This application claims priority to U.S. Provisional Application Ser.No. 60/502,878, filed Sep. 12, 2003, which is incorporated herein byreference in its entirety.

1. INTRODUCTION

The present invention relates generally to the field of hemodialysis,including methods and kits that can be employed to improve hemodialysistherapy. More specifically, the present invention relates to methods forreducing short-term and/or long-term vascular access complicationsassociated with hemodialysis therapy, for example reducing the averagenumber of vascular access interactions associated with a hemodialysistherapy, decreasing an infection rate associated with hemodialysistherapy, or preserving access to a hemodialysis vascular access site byreducing the time of compression applied to a vascular access site toabout one to about fourteen minutes, for the majority of hemodialysissessions. The present invention also provides for a kit comprising acomposition and instructions for achieving a reduction in vascularaccess complications associated with hemodialysis therapy for example, areduction in the average number of intreventions to replace or repairvascular access sites, a decrease in infection rate associated withhemodialysis therapy, or a preservation of access to a hemodialysisvascular access site.

2. BACKGROUND

Maintenance hemodialysis for end stage renal disease (ESRD) patientsrequires a reliable means of repetitive access to large blood vesselsthat are capable of rendering rapid extracorporeal blood flow to anartificial kidney. Typically an artery and vein are sutured to form afistulae which enlarges to a point of maturity over several months.Synthetic grafts are also used.

Hemodialysis patients receiving dialysis treatment via native veinarteriovenous fistulae and synthetic grafts typically undergo punctureof skin, subcutaneous tissue, and vascular access with 14-17 gaugeneedles two to three times weekly. When the procedure is finished andthe needles are removed from the skin, many patients bleed from thepuncture site for an extended period of time such that the standardtreatment involves post-hemodialysis compression at the site for atleast 15-20 minutes.

Problems are commonly associated with repeated vascular access, i.e.,access to circulation, and include hyperplasia, thrombosis, hematoma,venous stenosis, arterial stenosis, vascular occlusion, infection, andmorbidity. Thrombosis, i.e., a blood clot, is the primary cause ofaccess failure responsible for 50% of cases in polytetrafluoroethylene(PTFE) grafts. In those situations where anatomic lesions can beidentified, the pathology has been found to be intimal hyperplasia.Other causes of vascular access complications include: venous orarterial stenosis and infection (Mayers 1992, ASAIO J. 38:113-115).These complications with vascular access sites lead to blocking ornarrowing of vascular access sites which in turn result in an increasedincidence of surgery to repair, replace, or create new vascular accesssites. Degradation of the vascular access site also results in areduction in the delivered dose of dialysis through the use of temporarycatheters or reduced blood flow (Hakim and Himmelfarb, 1998, KidneyInternational, 54:1029-1040). Schwab found that 30% of hemodialysispatients with A-V fistulae, allowed to mature for 60 days, requiredintervention after about 800 days of hemodialysis therapy, and 80% ofhemodialysis patients with A-V grafts required intervention after about800 days of hemodialysis therapy. After 400 days of hemodialysistherapy, about 18% of A-V fistulae patients and 50% of A-V graftpatients required intervention, and after about 200 days 10% of A-Vfistulae and 30% of A-V graft patients required intervention (Schwab,1999, Kidney International, 55:2078-2090).

Hemodialysis vascular access is also a major risk factor for infectionand bacteremia, caused mostly by staphylococcal organisms, such as, butnot limited to, S. taphylococcus aureus and Enterococcus spp. (Nassarand Ayus, 2001, Kidney International 60:1-13; Tokars et al., 2002, AJIC30:288-295). These infections and bacteremia lead to complications suchas degradation in vascular access sites and surgical replacement ofvascular access sites. Other complications can include infectiousendocarditis, septic arthritis, epidural abscess, septic pulmonaryemboli, and osteomyelitis. Infections and bacteremia can be clinicallydiagnosed or a leukocyte-labeled indium scan of the vascular access sitecan be performed to identify infection where clinical manifestation ofinfection is not apparent or definite. One skilled in the art would knowhow to perform such scans and identify infections or resulting vascularaccess complications.

The pathology and risk factors for vascular access complications havebeen studied. Age, diabetes, the use of synthetic grafts, serum levelsof liproprotein(a) (Lp(a))≧57 mg/dL, serum fibronectin, calcification,apolipoprotein(a) serum levels, excessive compression of the vascularaccess site following hemodialysis or during sleep, turbulent blood flowand reduced blood pressure have been identified as predisposing toaccess occlusion (Berkoben, 1995, ANNA J. 22:17-24; Butterly, 1994, Adv.Ren Rep. Thpy. 1:163-166; Goldwasser 1994, AJKD 24:785-794; Astor etal., 2002, Kidney International 61:1115-1123). The cause of vascularaccess complications is suggested to be multifactorial and poorlyunderstood (Goldwasser, 1994, AJKD 24:785-794; Schwab, 1989, KidneyInternational, 36:707-711; and Windus, 1997, AJKD 29(4):560-564).

Hemodialysis patients also have an increased bleeding tendency due toplatelet dysfunction and ineffective platelet-vessel interaction inducedby uremia. In hemodialysis patients, the risk of prolonged bleeding isfurther increased by systematic anticoagulation resulting from thecontinuous infusion of heparin during the dialysis procedure (Di Minnoet al., 1985, Am. J. Med. 79:552-559). Many of these patients have ahigh incidence of cardiovascular risk factors. A retrospective studydone by the Department of Anesthesiology of the Mayo Clinic reportedthat for all the patients requiring creation of an A-V fistula in theyears 1986 to 1991, 92% suffered from hypertension, 86% from coronaryartery disease and 42% from a previous myocardial infarction(Solomonson, 1994, Anesth. Analg. 79:694-700). Most of this group ofpatients must be on prophylactic anticoagulation therapy with aspirin orwarfarin. Further, thiazide diuretics, which are commonly used to treathypertension or congestive heart failure, impair megakaryocyteproduction and can produce mild thrombocytopenia which may persist forseveral months after the drug is discontinued (Harrison's Principles ofInternal Medicine 13^(th) Ed. 1994, p. 1799).

Hemostasis, i.e., the stopping or cessation of bleeding, is oftencompromised in hemodialysis patients. The abnormal hemostasis associatedwith ESRD patients is most apparent in the prolonged post treatmentbleeding. Traditionally, when the cannulation needles are removed at theend of treatment, many hemodialysis patients require long compressiontimes at the site of removal to stop bleeding. Hemostasis is typicallyobtained by 15-20 minutes of manual compression (Schwab, 1994, KidneyInternational 36:707-711). Vaziri reported that in the population ofheparinizied ESDR patients studied the mean bleeding time was about 7-9minutes following hemodialysis which was reduced to about 3.2-3.3minutes with the topical administration of bovine thrombin to the sitewhere the hemodialysis needles are removed (Varizi et al., 1978, Journalof Dialysis, 2:393-398; and Varizi, 1979, Nephron 24:254-256).

Several compositions that can act as hemostatic agents and typicallyinclude collagen or fibrin are known (Falstrom et al., 1997,Catheterization and Cardiovasular Diagnosis 41:79-84; Hoekstra et al.,1998, Biomaterials. 19:1467-1471; Prior et al., 2000, Journal ofBiomedical Materials Research. 53(3):252-257). U.S. Pat. No. 4,394,373,for example, discloses compositions that act as coagulants and may beused to promote clotting of a wound by placing the compositions incontact with the wound where the composition comprises liquid or powderchitosan. U.S. Pat. No. 5,510,102, for example, discloses compositionsthat act as coagulants and may be used to promote clotting of a wound byplacing the compositions in contact with the wound where the compositioncomprises platelet rich plasma plus a biocompatible polymer that is ahemostatic agent such as alginate. The compositions of U.S. Pat. Nos.4,394,373 and 5,510,102 are either applied directly to the woundsurface, in the case of treatment of a superficial wound or in the caseof a puncture in an artery left by a needle or catheter.

Preserving access function and long-term vascular access is essentialfor the care of dialysis patients, particularly now that high-efficiencydialysis places even more demands on access function, and withincreasing numbers of older, sicker patients entering the ESRD programwith limited access sites. Vascular access complications remain thesingle greatest cause of morbidity and account for approximately onethird of all admissions and hospitalization days in the hemodialysispopulation (Spergel, 1997, Neph. News and Issues. 3:26-27, 35). Anaverage 1.2-2.8 surgical procedures per patient are performed each yearto repair or replace the vascular access site (Brothers et al., 1996, J.Sur. Research 60:312-316; Harland, 1994, Adv. Ren. Rep Therapy1:99-106). As much as $1 billion annually is spent on placement andmaintenance of vascular access (Spergel, 1997; Hakim and Himmelfarb,1998, Kidney International, 54:1029-1040).

High rates of vascular access complications in the hemodialysispopulation coupled with the increased use of high flux dialyzers, whichrequire higher blood flow, draw attention to the need for new methodsfor maintaining blood flow through vascular access sites (Hakim andHimmelfarb, 1998, Kidney International, 54:1029-1040).

The patents and published articles referenced in this section areincorporated by reference herein in their entirety.

3. SUMMARY

The advantages of the methods of the invention over the existingtraditional methods include, for example, the reduction in short-termand/or long-term vascular access complications, a reduction in thenumber of interventions to repair or replace a vascular access site, andthe increase in the length of time a vascular access site remains viablefor hemodialysis therapy.

In one aspect, the invention provides for a method for reducing vascularaccess complications associated with a hemodialysis therapy in a patientcomprising applying topically to a hemodialysis vascular access siteduring a hemodialysis session a composition comprising an amount of avasoconstrictor or coagulant effective for reducing or ceasingpost-hemodialysis bleeding at the vascular access site, and applyingcompression to the hemodialysis vascular access site for a period ofabout one to about fourteen minutes. In another aspect, the inventionprovides for a method for reducing vascular access complicationsassociated with a hemodialysis therapy in a patient comprising applyingtopically to a hemodialysis vascular access site during a hemodialysissession a composition comprising a barrier-forming material effectivefor reducing or ceasing post-hemodialysis bleeding in comparison togauze alone at the vascular access site, and applying compression to thehemodialysis vascular access site for a period of about one to fourteenminutes. In another aspect, the invention provides for a method forreducing the average number of interventions to repair or replacevascular access sites in a hemodialysis patient comprising applyingtopically to a hemodialysis vascular access site during a hemodialysissession a composition comprising an amount of a vasoconstrictor orcoagulant effective for reducing or ceasing post-hemodialysis bleedingat the vascular access site, and applying compression to thehemodialysis vascular access site for a period of about one to fourteenminutes. In another aspect, the invention provides for a method fordecreasing an infection rate associated with a hemodialysis therapy in apatient comprising applying topically to a hemodialysis vascular accesssite during a hemodialysis session a composition comprising an amount ofa vasoconstrictor or coagulant effective for reducing post-hemodialysisbleeding at the vascular access site, and applying compression to thehemodialysis vascular access site for a period of about one to fourteenminutes. In another aspect, the invention provides for a method forpreserving access to a hemodialysis vascular access site associated witha hemodialysis therapy in a patient comprising applying topically to ahemodialysis vascular access site during a hemodialysis session acomposition comprising an amount of a vasoconstrictor or coagulanteffective for reducing or ceasing post-hemodialysis bleeding at thevascular access site, and applying compression to the hemodialysisvascular access site for a period of about one to fourteen minutes.

In certain embodiments of the methods of the invention, compression isapplied to the vascular access site for a period of about one to aboutfourteen minutes for multiple sessions of the hemodialysis sessions ofthe therapy, preferably for the majority of hemodialysis sessions of thetherapy, and the number of vascular access complications associated withthe hemodialysis therapy is reduced in comparison to hemodialysispatients for whom compression is applied for a period of greater thanabout fourteen minutes for the majority of a comparable number ofhemodialysis sessions. In certain embodiments of the methods of theinvention, the vascular access complications are long-term, such as, butnot limited to, hyperplasia, thrombosis, venous stenosis, arterialstenosis and/or morbidity. In certain embodiments, the vascular accesscomplications are short-term, such as, but not limited to, hematomasand/or infections.

In certain embodiments, the composition comprises a coagulant. In otherembodiments, the composition comprises a vasoconstrictor. In yet otherembodiments, the composition comprises a coagulant and avasoconstrictor.

In certain embodiments, the vascular access complication reduced orceased by the methods and/or kits of the invention is hyperplasia,thrombosis, hematoma, venous stenosis, arterial stenosis, or infection.In particular embodiments, failure of vascular access sites due tothrombosis is reduced to fewer than 80% of patients having vascularaccess complications. In particular embodiments, failure of vascularaccess sites due to venous stenosis is reduced to fewer than 80% ofpatients having vascular access complications. In particularembodiments, failure of vascular access sites due to arterial stenosisis reduced to fewer than 2% of patients having vascular accesscomplications. In particular embodiments, failure of vascular accesssites due to infection is reduced to fewer than 15% of patients havingvascular access complications. In other embodiments, failure of vascularaccess sites due to infection is reduced to fewer than 20% of patientshaving vascular access complications. In certain embodiments of themethods and kits of the invention where the patient has a mature A-Vfistulae, vascular access complications are reduced by about 10% innumber or severity after about 200 days of hemodialysis therapy by about20% in number or severity after about 500 days of hemodialysis therapy,or by about 30% in number or severity after about 800 days of therapy,relative to compression without coagulant or vasoconstrictor (e.g.,compression with gauze alone). In certain embodiments of the methods andkits of the invention where the patient has a mature A-V graft, vascularaccess complications are reduced to by about 20% in number or severityafter about 100 days of hemodialysis therapy, by about 35% in number orseverity after about 200 days of hemodialysis therapy, or by about 50%in number or severity after about 500 days of therapy, relative tocompression without coagulant or vasoconstrictor (e.g., compression withgauze alone).

In certain embodiments of the methods and kits of the invention, themethods of the invention further comprise the proviso that the patientis not concurrently treated with erythropoietin. The erythropoietin maybe EPOGEN™. In embodiments where the patient is not concurrently treatedwith erythropoietin, this means that the patient has not receivederythropoietin for preferably about 1 week. 2 weeks, 3 weeks, 4 weeks,1.5 months, 2 months, 2.5 months, 3 months, 3.5 months, 4 months, 5,months, 6 months, 7 months, 8 months, 9 months, 10 months 11 months or12 months prior to initiation of therapeutic regimens of the invention.In certain embodiments of the methods and kits of the invention, whereinerythropoietin is administered to the patient in conjunction withpracticing the methods of the invention, the amount of erythropoietin isreduced in comparison to hemodialysis patients for whom compression isapplied for a period of greater than about fourteen minutes for themajority of hemodialysis sessions. In related embodiments of the methodsand kits of the invention, the erythropoietin administered comprisesless than about 3,000 units per dose, or as a total of multiple doses,of recombinant erythropoietin or epoetin alfa.

In certain embodiments of the methods and kits of the invention, thevascular access complication is reduced blood flow through thehemodialysis vascular access site relative to a newly matured vascularaccess site. In related embodiments, the blood flow is measured in thenewly matured vascular access site prior to any hemodialysis use. Inrelated embodiments, the blood flow is measured in the newly maturedvascular access site in the patient prior to any hemodialysis use. Incertain embodiments of the methods and kits of the invention, thevascular access complication is reduced blood flow through thehemodialysis vascular access site relative to the average blood flowthrough a newly matured vascular access site. In one embodiment, theaverage blood flow is the average flow for newly matured vascular accesssites in ESRD patients.

In another aspect, the invention provides for a method for reducingvascular access complications associated with a hemodialysis therapy ina patient comprising applying topically to a hemodialysis vascularaccess site during a hemodialysis session a composition comprising abarrier-forming material effective for reducing or ceasingpost-hemodialysis bleeding at the vascular access site in comparison togauze alone; and applying compression to the hemodialysis vascularaccess site for a period of about one to about fourteen minutes, whereinsuch compression is applied to the hemodialysis vascular access site fora period of about one to about fourteen minutes for multiple sessions ofthe hemodialysis sessions of the therapy, preferably for the majority ofhemodialysis sessions of the therapy, and the number of vascular accesscomplications associated with the hemodialysis therapy is reduced incomparison to hemodialysis patients for whom compression is applied fora period of greater than about fourteen minutes for the majority ofhemodialysis sessions. In certain embodiments, the composition comprisesa coagulant. In other embodiments, the composition comprises avasoconstrictor. In yet other embodiments, the composition comprises acoagulant and a vasoconstrictor. In yet other embodiments, thecomposition comprises collagen. In still other embodiments, thecomposition of the method and kits of the invention comprises avasconstrictor, coagulant, and/or barrier-forming material, with theproviso that the composition does not comprise collagen.

In yet another aspect, the invention provides for a method for reducingthe average number of interventions to surgically repair or replacevascular access sites in a hemodialysis patient comprising applyingtopically to a hemodialysis vascular access site during a hemodialysissession a composition comprising an amount of a vasoconstrictor orcoagulant effective for reducing or ceasing post-hemodialysis bleedingat the vascular access site; and applying compression to thehemodialysis vascular access site for a period of about one to fourteenminutes, wherein such compression is applied for about one to aboutfourteen minutes for multiple sessions of the hemodialysis sessions ofthe therapy, preferably for the majority of hemodialysis sessions of ahemodialysis therapy, and the average number of interventions to replaceor repair vascular access sites is reduced in comparison to hemodialysispatients for whom compression is applied for a period greater than aboutfourteen minutes for the majority of hemodialysis sessions. In certainembodiments, the composition comprises a coagulant. In otherembodiments, the composition comprises a vasoconstrictor. In yet otherembodiments, the composition comprises a coagulant and avasoconstrictor.

In embodiments of the methods and kits of the invention, theintervention comprises surgery to repair or replace a vascular accesssite. In one embodiment, the average number of interventions is fewerthan about 2.8 per year. In another embodiment, the average number ofinterventions is fewer than about 2.0 per year. In yet anotherembodiment, the average number of interventions is fewer than about 1.2per year.

In another aspect, the invention also provides for a method fordecreasing an infection rate associated with a hemodialysis therapy in apatient comprising applying topically to a hemodialysis vascular accesssite during a hemodialysis session a composition comprising an amount ofa vasoconstrictor or coagulant effective for reducing post-hemodialysisbleeding at the vascular access site; and applying compression to thehemodialysis vascular access site for a period of about one to fourteenminutes, wherein such compression is applied for about one to aboutfourteen minutes for multiple sessions of the hemodialysis sessions ofthe therapy, preferably for the majority of hemodialysis sessions of thetherapy, and the rate of infection associated with hemodialysisdecreases in comparison to hemodialysis treatments in patients for whomcompression is applied for a period greater than about fourteen minutesfor the majority of hemodialysis sessions. In certain embodiments, thecomposition comprises a coagulant. In other embodiments, the compositioncomprises a vasoconstrictor. In yet other embodiments, the compositioncomprises a coagulant and a vasoconstrictor. In certain embodiments, thevascular access complications due to infection reduced by about 15% innumber or severity, relative to compression without coagulant orvasoconstrictor (e.g., compression with gauze alone). According tocertain aspects of the invention, the infection is HIV, Hepatitis C,macrolides, lincomycin, vancomycin resistant enterococcus, orstreptogramin B (MLS resistance). In one aspect of the invention, thecomposition further comprises an anti-fungal, anti-viral, orantibacterial agent.

The invention further provides for a method for preserving access to ahemodialysis vascular access site associated with a hemodialysis therapyin a patient comprising applying topically to a hemodialysis vascularaccess site during a hemodialysis session a composition comprising anamount of a vasoconstrictor or coagulant effective for reducing orceasing post-hemodialysis bleeding at the vascular access site; andapplying compression to the hemodialysis vascular access site for aperiod of about one to about fourteen minutes, wherein such cessationcompression is applied for about one to about fourteen minutes formultiple sessions of the hemodialysis sessions of the therapy,preferably for the majority of hemodialysis sessions of the therapy, andthe access to the hemodialysis vascular access site is preserved incomparison to hemodialysis patients for whom compression is applied fora period greater than about fourteen minutes for the majority ofhemodialysis sessions. In certain embodiments, the composition comprisesa coagulant. In other embodiments, the composition comprises avasoconstrictor. In yet other embodiments, the composition comprises acoagulant and a vasoconstrictor.

In certain embodiments of the invention, an effective amount of avasoconstrictor and/or coagulant is an amount that results in reductionor cessation of bleeding when applied to a catheter exits wound withoutcompression. In other embodiments of the invention, an effective amountof a vasoconstrictor and/or coagulant is an amount that results inreduction or cessation of bleeding when applied to a catheter exitswound with compression according to the methods of the invention.

In certain embodiments, the methods of the invention are practiced inthe first hemodialysis session the patient undergoes. In relatedembodiments, the methods of the invention are practiced in all of thehemodialysis session the patient undergoes. In still other embodiments,the patient has not had any hemodialysis sessions prior to initiating atherapeutic regimen of the invention. In still other embodiments, thepatient has had about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 45, or 50 hemodialysis sessions where the methods of theinvention were not used or compression was applied for about 10, 11, 12,13, or 14 minutes or greater. In certain embodiments, the methods of theinvention are practiced on the patient for about 3 months, 6 months, 1year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, 5 years, 6 years,7 years, 8 years, 9 years, 10 years, 11 years, or 15 years.

In certain embodiments of the methods and kits of the inventiondescribed herein, the therapeutic regimens of the invention are at leastten months in duration.

In certain embodiments of the methods and kits of the inventiondescribed herein, the vasoconstrictor or coagulant is substantially freeof allergens or is non-immunogenic. In related embodiments, thevasoconstrictor or coagulant does not comprise bovine derived thrombin.

In preferred embodiments of the methods and kits of the inventiondescribed herein, the therapeutic regimen of the invention entailsmaintaining compression for about one to five minutes in conjunctionwith topical application of a vasoconstrictor or coagulant and thisprocedure is preferably repeated, preferably for the majority ofhemodialysis sessions of the therapy. In another preferred embodiment ofthe methods and kits of the invention described herein, the compressionis maintained for about one to ten minutes for the majority ofhemodialysis sessions of the therapy.

In certain embodiments of the methods and kits of the inventiondescribed herein, the coagulant comprises alpha-2-antiplasmin,alpha-1-antitrypsin, alpha-2-macroglobulin, aminohexanoic acid,aprotinin, a source of calcium ions, calcium alginate, calcium-sodiumalginate, casein kinase II, chitin, chitosan, collagen, cyanoacrylates,epsilon-aminocaproic acid, Factor XIII, fibrin, fibrin glue, fibrinogen,fibronectin, gelatin, living platelets, metha-crylates, PAI-1, PAI-2,plasmin activator inhibitor, p-GlcNAc, plasminogen, platelet agonists,protamine sulfate, prothrombin, an RGD peptide, sphingosine, asphingosine derivative, thrombin, thromboplastin, or tranexamic acid.

In certain embodiments of the methods and kits of the inventiondescribed herein, the vasoconstrictor is adrenaline, endothelin-1,epinephrine, phenylephrine, serotonin, thromboxane, or U-46619.

In certain embodiments of the methods and kits of the inventiondescribed herein, the composition further comprises collagen.

In one aspect of the methods and kits of the invention described herein,the composition further comprises a pharmaceutical carrier.

In certain embodiments of the methods and kits of the inventiondescribed herein, the hemodialysis vascular access site comprises a veinsutured to an artery. In related embodiments of the methods and kits ofthe invention described herein, the hemodialysis vascular access sitecomprises a native arteriovenous fistula. In other embodiments of themethods and kits of the invention described herein, the hemodialysisvascular access site comprises a synthetic vascular graft.

In certain embodiments of the methods and kits of the inventiondescribed herein, the therapeutic regimens of the invention arepracticed two or three times per week.

In certain embodiments of the methods of the invention described herein,before applying topically to a hemodialysis vascular access site duringa hemodialysis session a composition of the invention in conjunctionwith compression, an anticoagulant is administered to the patient. Inrelated embodiments of the methods of the invention described herein,the anticoagulant is selected from the group consisting of coumadin,heparin, nadroparin, asparin, and a thrombolytic agent. In yet otherrelated embodiments of the methods of the invention described herein,the composition further comprises protamine sulfate in an amounteffective to neutralize heparin.

In certain embodiments of the methods of the invention described herein,the compression is manual compression. In related embodiments of themethods of the invention described herein, the compression is notocclusive and blood flow continues in the vascular access site. In otherembodiments of the methods of the invention described herein, thecompression is mechanical compression. In certain embodiments of themethods of the invention described herein, the compression is applied toa vein or artery proximal of the hemodialysis vascular access site. Inembodiments of the methods of the invention described herein, thecompression is applied with a compression bandage.

According to one aspect of the methods of the invention describedherein, the composition is formulated as a gel, solid, liquid, sponge,foam, spray, emulsion, suspension, film, membrane, mat, string,microbead, microsphere, microfibril, or solution. In certain embodimentsof the methods of the invention described herein, the compositionfurther comprises a neutral liquid, neutral gel or neutral solid. Inpreferred embodiments the neutral solid comprises a gauze, a bandage, ora barrier-forming material. In other preferred embodiments, the neutralsolid is a gauze. In related embodiments of the methods of the inventiondescribed herein, the film, membrane, or mat comprises a barrier-formingmaterial. In certain embodiments of the methods of the inventiondescribed herein, the composition is in the form of a coating on theneutral solid.

In certain embodiments of the methods of the invention described herein,the hemodialysis is administered using lumen canulation needles that arefewer than 15 gauge.

In certain embodiments of the methods of the invention described herein,the patient is a human. In other embodiments, the patient is diabetic.In other embodiments, the patient has hypertension. In yet other relatedembodiments, the patient has serum fibronectin. In still otherembodiments, the patient has reduced blood pressure relative to theaverage pressure for a healthy individual comparable in age, race,gender, and/or stage of renal disease. In still other embodiments, thepatient is greater than 55 years in age. In still other embodiments, thepatient is an end stage renal disease patient. In still otherembodiments, the patient is in need of hemodialysis but is not an endstage renal disease patient. In still other embodiments, the patient ismale. In still other embodiments, the patient is female. In still otherembodiments, the patient is African American.

In certain embodiments of the methods of the invention, the patient hasan average hemostasis time of about (a) 12 to 15, (b) 15 to 18, (c) 18to 21, or (d) 21 to 24 minutes using compression alone. In certainembodiments of the methods of the invention, the patient has an averagehemostasis time greater than 11 minutes using compression alone. Incertain embodiments of the methods of the invention, the compression isapplied with a vasoconstrictor and/or coagulant for (a) fewer than 11minutes, (b) about 9 to 10 minutes, (c) about 8 to 9 minutes, or (d)about 7 to 8 minutes.

According to another aspect of the invention, the invention provides fora kit comprising in one or more containers a composition comprising anamount of a vasoconstrictor and/or coagulant effective for reducing orceasing post-hemodialysis bleeding; and a pharmaceutically acceptablecarrier, and instructions for practicing the methods of the invention.In certain embodiments, the instructions provide for topically applyingthe composition at a hemodialysis vascular access site where compressionis applied for about one to fourteen minutes, resulting in a reductionin vascular access complications.

The invention yet further provides for a method for reducing bleedingfrom a hemodialysis vascular access site, comprising applying topicallyas part of a hemodialysis therapy to a hemodialysis vascular access siteduring a hemodialysis session a composition comprising an amount of avasoconstrictor or coagulant effective for reducing or ceasingpost-hemodialysis bleeding at the vascular access site; and applyingcompression to the hemodialysis vascular access site for a periodsufficient to achieve cessation or reduction of blood flow out of thehemodialysis vascular access site, such that a cessation or reduction ofbleeding from the hemodialysis vascular access site is achieved in aboutone to fourteen minutes, for the majority of hemodialysis sessions ofthe therapy.

3.1. Terminology

The term “reduce” (in reference to blood flow) means that blood flowfrom a vascular access site has decreased to a rate or amount that isacceptable such that the patient is not in need of supervision and canleave the hemodialyis facility.

The term “hemodialysis session” means the period of time beginning whenhemodialysis needles are inserted into a vascular access site of apatient that will undergo hemodialysis and includes the timehemodialysis is performed and bleeding following removal of hemodialysisneedles up to time hemostasis is reached and compression is no longerneeded. Hemodialysis therapy comprises at least four hemodialysissessions or is for at least one week. In certain embodiments,hemodialysis therapy is measured from the beginning of use of a newlymatured vascular access site. In certain other embodiments, hemodialysistherapy is measured from the beginning of the use of the methods of theinvention.

The term “vascular access site” means: the surgically created vascularconnection between an artery and vein in addition to the contiguousenlarged portion of the connected blood vessels; the surgicallyimplanted synthetic grafts connecting blood vessels; or the site ofinsertion or removal of hemodialysis needles.

The term “vascular access complication” means any cause that reducesblood flow through the hemodialysis vascular access site including, forexample: ischaemic vascular disease, hyperplasia, intimal hyperplasia,thrombosis, hematoma, venous stenosis, arterial stenosis, or infection.Blood flow through the vascular access site, measured when the patientis at rest and no compression is being applied, is reduced in comparisonto blood flow through the vascular access site, measured when thepatient is at rest and no compression is being applied, at an earliertime, prior to multiple hemodialysis sessions, or when the site hasmatured but has not yet been used for hemodialysis.

The term “about” means ±10% of the value the term to which the term isapplied.

4. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and kits useful for reducingvascular access complications associated with hemodialysis therapy andprolonging the period of time at vascular access site can be used in apatient. Hemostasis is typically obtained by a least 15-20 minutes ofmanual compression following hemodialysis. While this time is aninconvenience to the patient, the experimentation and results of theApplicants' demonstrate that such a prolonged compression time is alsothe cause of vascular access complications. The present invention isbased in part on the inventors' determination that a reduction in thecompression time to a period of about one to fourteen minutes, repeated,preferably, for the majority of hemodialysis sessions results in areduction in, prevention, or inhibition of, vascular accesscomplications.

Without being bound by any particular mechanism by which the methods ofthe invention achieve the desired goals, inventors believe thatprolonged stasis of blood through the portion of the vascular accesssite where the artery and vein are connected or where the syntheticgraft is located causes complications such as, but not limited to,clotting, stenosis, or hyperplasia. These complications result in areduction in blood flow through the vascular access site in the absenceof compression which decreases the efficiency or ability to effectivelyperform hemodialysis. The site where the hemodialysis needles areremoved, i.e., the site where compression is typically applied, maydiffer from the location in the vascular access site as the A-V fistulaconnection or graft, since vascular access sites can be, for example,brachial cephalic or radial cephalic.

In one aspect, methods for reducing vascular access complications of thepresent invention comprise topical administration of compositionscomprising a vasoconstrictor and/or coagulant. In another aspect,methods for reducing vascular access complications of the presentinvention comprise topical administration of a barrier-forming material.Presented below, is, first, a description of compositions suitable foruse in conjunction with the methods of the invention and, second, adescription of the methods and kits of the invention.

4.1. Compositions for Use in the Methods of the Invention

The compositions for use in the methods and kits of the invention cancomprise a barrier-forming material for topical administration. Suchbarrier-forming materials can, for example, comprise a vasoconstrictorand/or a coagulant. In addition, the compositions for use in the methodsand kits of the invention can comprise a vasoconstrictor and/or acoagulant in an amount effective for reducing or ceasingpost-hemodialysis bleeding at a vascular access site. The compositionsmay contain other components, for example, anti-bacterial agents,anti-viral, or anti-fungal agents to prevent infection associated withhemodialysis. Various formulations of the compositions can be used inthe methods and kits of the invention, including, but not limited to,solid compositions, gels, foams, or liquids that can be appliedtopically to a vascular access site. The compositions for use in themethods and kits of the invention reduce the compression needed to stopbleeding from a hemodialysis vascular access site. The reduction incompression, to about one to fourteen minutes, more preferably to one toeleven minutes, and more preferably to one to ten minutes, perhemodialysis session, results in a reduction in vascular accesscomplications, relative to compression without coagulant orvasoconstrictor (e.g., compression with gauze alone).

In addition to the above, the present invention also encompasses use ofthe methods of Examples 1 and 2 in conjunction with compositions thatwill further reduce the period of time required to achieve hemostasis inthe hemodialysis patient population. Such compositions may comprise, butare not limited to, one or more vasoconstrictors, coagulants,anti-inflammatory compounds, and combinations thereof, that aretopically administered to the vascular access site. In particularinstances, such compositions are topically applied as part of acomposition comprising a barrier-forming material.

4.1.1. Vasoconstrictors

In one embodiment, the composition for use in the methods and kits ofthe invention may include one or more vasoconstrictors. Thevasoconstrictor(s), for example, can be one or more of the following:endothelin-1, epinephrine, phenylephrine, serotonin, thromboxane,norepinephrine, prostaglandin, methergine, oxytocin, or isoprelandU-46619 (Cayman Chemical, Ann Arbor, Mich.; a stable prostaglandinendoperoxide analog which serves as a thromboxane mimetic).

The vasoconstrictors may be used in standard recommended dosages, or, incertain embodiments, in dosages of no greater than approximately 30%,50%, 70%, 80%, or 90% of the standard recommended dosage.

4.1.2. Coagulants

In one embodiment, the composition may include one or more coagulants.The coagulant(s), for example, can be one or more of the following:alpha-2-antiplasmin, alpha-1-antitrypsin, alpha-2-macroglobulin,aminohexanoic acid, aprotinin, beta₂-glycoprotein I, a source of calciumions, calcium alginate, calcium-sodium alginate, casein kinase II,chitin, chitosan, collagen, cyanoacrylates, epsilon-aminocaproic acid,Factor X, Factor IX, Factor X, Factor XIII, fibrin, fibrin glue,fibrinogen, fibronectin, gelatin, living platelets, metha crylates,PAI-1, PAI-2, p-GlcNAc, plasmin activator inhibitor, plasminogen,platelet agonists, protamine sulfate, protein C, prothrombin, an RGDpeptide, sphingosine, a sphingosine derivative, thrombin,thromboplastin, or tranexamic acid. In one embodiment, the compositionfor use in the methods and kits of the invention does not comprise acoagulant. In another embodiment, the composition does not comprisethrombin. In a related embodiment, the composition does not comprisebovine thrombin.

The coagulatns may be used in standard recommended dosages, or, incertain embodiments, in dosages of no greater than approximately 30%,50%, 70%, 80%, or 90% of the standard recommended dosage.

4.1.3. Other Components

The compositions for use in the methods and kits of the presentinvention can comprise additional components. For example, thecompositions may contain other components, for example, anti-bacterialagents, anti-viral, or anti-fungal agents to prevent infectionassociated with hemodialysis. The compositions for use in the methodsand kits of the invention can also comprise wound-healing and/orpain-reducing agents. Such agents include anti-inflammatory agents, bothsteroidal and non-steroidal, such as, but not limited to, agents whichinhibit leukocyte migration into the area of the puncture in the bloodvessel or graft of the vascular access site (e.g., silver sulfadiazinemacetylsalicylic acid, indomethacin, and Nafazatrom), anti-histamines(e.g., pyrilamine, chlorpheniramine, tetraydrozoline, antazoline,cortisone, hydrocortisone, beta-methasone, dexamethasone, fluocortolone,prednisolone, triamcinolone, indomethacin, and sulindac, its salts andits corresponding sulfide); agents which inhibit free radical formation(e.g., superoxide dismutase (SOD), catalase, glutathione peroxidase,b-carotene, ascorbic acid, transferring, ferritin, ceruloplasmin, anddesferrioxamine alpha-tocophenol); and bacteriostatic agents orbacteriocidal agents, (e.g., cefoxitin, n-formamidoyl thienamycin,tetracyclines, chloramphenicol, neomycin, gramicidin, bacitracin,sulfonamides, oxacillin, gentamycin, gentamycin, mupirocin, kanamycin,vancomyacin, amikacin, sisomicin, silver, silver tobramycin,norfloxican, nitrofurazones, and the combination offluoroalanin/pentizidone).

The compositions for use in the methods and kits of the invention canalso include a pharmaceutically acceptable carrier such as, but notlimited to, conventional excipients, e.g., pharmaceutically acceptableorganic or inorganic carrier substances suitable for topical applicationwhich do not deleteriously react with the compositions of the invention.Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt, sugar solutions, etc. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents.

In one embodiment, the composition for use in the methods and kits ofthe invention does not comprise an agent that has allergenic properties.Vasoconstrictors and/or coagulant agents as well as pharmaceuticalcarriers and other components can easily be tested for allergicactivities by methods known to those of skill in the art. For example,delayed hypersensitivity skin tests are of great value in determiningallergenic reactions to agents in hemodialysis patients or in animalmodels (Sato et al., 1995, Clin. Immunol. Pathol. 74:35-43).

Erythropoietin, epoetin alfa recombinant epogen, or Epogen™, has 3,000units of recombinant erythropoietin, 2.5 mg albumin (human) in sterilebuffered solution (pH 6.9+/−0.3) of sodium citrate (5.8 mg), sodiumchloride (5.8 mg), citric acid (0.06 mg), in water for injection. Usesof erythropoietin include stimulating the body to make red blood cells.Loss of blood cells due to excessive and repeated bleeding can lead todecreased hematocrit levels, anemia, and other complications (Eschbach,2002, J. Am. Soc. Nephrol. 13:1412-1414). Hemodialysis patients canexhibit decreased hematocrit levels. The methods of the invention resultin a reduction in bleeding associated with hemodialysis, a reduction ininterventions due to vascular access complications, and/or a reductionin vascular access complications, which in turn reduces the need forerythropoietin. In one embodiment, the methods of the invention thepatient is not concurrently treated with erythropoietin. In oneembodiment, wherein erythropoietin is administered to the patient, theamount of erythropoietin is reduced in comparison to hemodialysispatients for whom compression is applied for a period of greater thanabout fourteen minutes for the majority of hemodialysis sessions.

4.1.4. POLY-β-1→4-N-ACETYLGLUCOSAMINE

Section 2 above incorporates by reference numerous U.S. Patent documentsthat describe in detail the structure of poly-β-1→4-N-acetylglucosamine,any of which can be used in the compositions used in the methods andkits of the invention.

In preferred embodiments, poly N-acetylglucosamine is derived from theprocess of a) treating a microalgae comprising a cell body and a polyN-acetylglucosamine with a agent, e.g., hydroflouric acid, capable ofweakening of the cell wall of a micoralga for a sufficient time so thatthe poly N-acetylglucosamine is released from the cell body; b)segregating the poly N-acetylglucosamine from the cell body; and c)removing all or, substantially all organic contaminants, andsubstantially all inorganic contaminants from the segregated polyN-acetylglucosamine, so that the poly N-acetylglucosamine species isisolated. The poly N-acetylglucosamine used in the methods of theinvention is preferably biocompatible and/or immunoneutral.

As used herein derivatives of a poly N-acetylglucosamine polymerinclude: a semi crystalline form of a poly N-acetylglucosamine polymer;a poly N-acetylglucosamine polymer comprising about 50 to about 150,000N acetylglucosamine monosaccharides covalently attached in a β-1→4conformation, and said polymer has a molecular weight of about 10,000daltons to about 30 million daltons; a poly β-1→4-acetylglucosaminepolymer comprising about 50 to about 50,000 N acetylglucosaminemonosaccharides covalently attached in a P-1→4 conformation, and saidpolymer has a molecular weight of about 10,000 daltons to about 10million daltons; a poly β-1→4-acetylglucosamine polymer comprises about50 to about 10,000 N acetylglucosamine monosaccharides covalentlyattached in a β-1→4 conformation, and said polymer has a molecularweight of about 10,000 daltons to about 2 million daltons; a polyβ-1→4-acetylglucosamine polymer comprising about 50 to about 4,000 Nacetylglucosamine monosaccharides covalently attached in a β-1→4conformation, and said polymer has a molecular weight of about 10,000daltons to about 800,000 daltons; and a semi crystalline poly β-1→4-Nacetylglucosamine polymer comprising at least one N acetylglucosaminemonosaccharide that is deacetylated, and wherein at least 40% of said Nacetylglucosamine monosaccharides are acetylated. Derivatives of a polyβ-1→4-N-acetylglucosamine polymer also include compositions that areabout 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less polyβ-1→4-N-acetylglucosamine. The poly-β-1→4-N-acetylglucosamine polymercan be purified. The poly-β-1→4-N-acetylglucosamine polymer can beacetylated. The poly-β-1→4-N-acetylglucosamine polymer can bedeacetylated. The poly-β-1→4-N-acetylglucosamine polymer can be free ofprotein, substantially free of other organic contaminants, andsubstantially free of inorganic contaminants. Thepoly-β-1→4-N-acetylglucosamine polymer can be semi-crystalline. Thepoly-β-1→4-N-acetylglucosamine polymer can be biodegradable andbiocompatible. The poly-β-1→4-N-acetylglucosamine polymer can have amolecular weight of about 800,000 daltons to about 30 million daltons.The poly-β-1→4-N-acetylglucosamine can comprise semi crystalline havinga molecular weight of about 800,000 daltons to about 30 million daltons.The poly-β-1→4-N-acetylglucosamine polymer can have a molecular weightof about 10,000 daltons to about 800,000 daltons. Thepoly-β-1→4-N-acetylglucosamine can comprise semi crystalline having amolecular weight of about 10,000 daltons to about 800,000 daltons. Inone embodiment, the methods and kits of the invention have the provisothat the composition does not comprise poly-P-1→4-N-acetylglucosamine ora derivative thereof.

4.1.5. Formulations of the Compositions of the Invention

The composition for use in the methods and kits of the invention can beformulated in any number of ways, including, but not limited to, a gel,solid, liquid, sponge, foam, spray, emulsion, suspension, solution,string, microbead, microsphere, or microfibril. The compositions caninclude, for example, a pharmaceutically acceptable carrier, a neutralliquid, neutral gel or neutral solid. In certain preferred embodiments,the composition is formulated as a barrier, membrane, or film. Moreover,the composition can be added to a barrier, membrane, or film, such as abacking. A barrier, membrane, or film can be supplied in a variety ofstandard sizes, which can be further cut or sized to the area beingtreated. The barrier, membrane, or film can be, for example, aconventional bandage or gauze to which the composition of the inventionis added or coated on, prior to application to the patient. In anotherembodiment, the composition can be administered topically followed byapplication of a barrier, membrane, or film. Alternatively, thecomposition can be, for example, formulated as a barrier, membrane, orfilm made out of, for example, strings, microbeads, microspheres, ormicrofibrils, or the composition can be formulated as a barrier-formingmat.

In certain embodiments, the composition is formulated as a gel. The gelcan be of varying viscosity. For embodiments where the gel is applied toa bandage to topically treat a site of needle puncture, a low viscosityis desired. For gels, higher viscosity may be desired if the compositionis intended to remain in a location rather than dissipate rapidly.Viscosity is the quantity that describes a fluid's resistance to flow,while the range of viscosity is a continuum. For example, as a frame ofreference, not as a limitation of the meaning of viscosity, theviscosity values of about 1→4 centipoise (cP) generally are typified byfluid compositions. Viscosity values of about 5-14 cP generally aretypified by gel-like compositions, while viscosity values of 15-20 cPare relatively hard compositions such as plastics. The viscosity of cellcytoplasm is about 11 cP. In certain embodiments, the viscosity of thecompositions for topical administration in the methods and kits of theinvention is about 10 cP. Viscosity can be measured with, for example, aSaybolt International B.V. (Vlaardingen, The Netherlands). One skilledin the art can also routinely use other measurement techniques anddevices common in the art.

In certain embodiments of the invention, the composition is formulatedas a membrane. In other embodiments, the composition is in a containerwithin or coated on a membrane. The membranes may be porous orrelatively continuous. In preferred embodiments the membranes are madeof woven polymer fibers. Such membranes are particularly useful intreatment of wounds on the skin surface.

In one embodiment, the composition for use in the methods and kits ofthe invention comprises, barrier-forming materials, for example, a patchmade of barrier-forming materials. In another embodiment, thecomposition comprises a material, e.g., a barrier-forming material,coated with a composition of the invention. In one such embodiment, thecomposition comprises a gauze coated with a composition of theinvention. In certain embodiments, the composition comprises abarrier-forming material coated with the composition of the invention,wherein the barrier-forming material also contains an adhesive so thatthe material can adhere to a patient's skin surface. Alternatively, thecomposition can lack barrier-forming materials.

In another embodiment, where the composition for use in the methods andkits of the invention encompasses an adhesive barrier-forming material,the composition can be topically adhered to cover a skin surface woundcaused by removal of hemodialysis needles that is contiguous with avascular access site without applying pressure to compress the vascularaccess site.

The compositions for use in the methods and kits of the invention caninclude a backing. For example, if the composition is formulated as apatch, a backing can be adhered to the patch. The backing can, forexample, be coated or embedded with any adhesive compound so that areasof the backing that contact the skin will adhere the backing and theattached composition of the invention to the skin surface of thepatient. The type of adhesive used can be any type of medicallyacceptable adhesive. Such backings can be made of natural polymers orsynthetic materials. Natural polymers from which the backing can be madeinclude, but are not limited to, cellulose and xylan. Syntheticmaterials from which the backing can be made include, but are notlimited to, polyurethane, Teflon, Dacron, stainless steel mesh screen,and a polyester woven fabric. Preferably the backing and adhesive areporous to areas which contact the skin to allow diffusion of oxygen. Thebacking can also serve as a surface upon which manual compression can beapplied.

4.2. Methods for Reducing Vascular Access Complications

In general, the invention relates to methods for reducing vascularaccess complications associated with a hemodialysis therapy in a patientcomprising applying topically to a hemodialysis vascular access siteduring a hemodialysis session a composition comprising an amount of avasoconstrictor or coagulant effective for reducing or ceasingpost-hemodialysis bleeding at the vascular access site, and applyingcompression to the hemodialysis vascular access site for a period ofabout one to fourteen minutes, so that compression is applied to thehemodialysis vascular access site for a period of about one to aboutfourteen minutes, for repeated hemodialysis sessions, preferably for themajority of hemodialysis sessions of the therapy and the number ofvascular access complications associated with the hemodialysis therapyis reduced in comparison to hemodialysis patients for whom compressionis applied for a period of greater than about fourteen minutes for themajority of hemodialysis sessions.

In certain embodiments of the methods of the invention, the method stepsare repeated at least about 4, 5, 6, 7, 8, 9, or 10 times. In certainembodiments of the methods of the invention, the methods include thestep of repeating the first two steps of applying composition andcompression at least about 4, 5, 6, 7, 8, 9, or 10 times. In certainembodiments, the methods of the invention are practiced for at leastabout 50% of the hemodialysis sessions of a hemodialysis therapyregimen. In certain embodiments, the methods of the invention arepracticed for at least about 70% of the hemodialysis sessions of ahemodialysis therapy regimen. The regimen is preferably at least oneweek, at least one month, at least two months, at least three months, orat least six months in duration, with anywhere from 0, 1, 2, 3, or morehemodialysis sessions per week. In other embodiments, the methods of theinvention are practiced during at least 50% of the hemodialysis sessionsover a six month period.

In certain embodiments, the hemodialysis regimens of the invention aregreater than one week in duration, wherein a patient typically receives1-3 hemodialysis sessions. For example, the hemodialysis regimens of theinvention may comprise at least 4, 5, 6, 7, 8, 9, or 10 hemodialysissessions. In certain embodiments, the hemodialysis regimens of theinvention are at least ten months in duration. In yet other embodiments,the hemodialysis regimens of the invention are at least about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30years in duration. In a preferred embodiment, the hemodialysis regimensof the invention is at least 5-15 years in duration. The methodsdescribed herein are preferably practiced during at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% of hemodialysis sessions. Thehemodialysis sessions of the invention are useful for prolonging the useof vascular access sites. Thus, the use of a particular vascular accesssite can be maintained or prolonged for at least about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 years asa result of practicing the methods described herein. In certainembodiments, the number of times a particular vascular access site isused is decreased in comparison to hemodialysis practiced without themethods of the invention, for example using compression with gauzealone. In other embodiments, the number of hemodialysis sessions neededby the patient is decreased in comparison to hemodialysis practicedwithout the methods of the invention, e.g., using compression with gauzealone.

In certain embodiments of the methods and kits of the invention, thevascular access complication is hyperplasia, thrombosis, hematoma,venous stenosis, arterial stenosis, infection, or morbidity. In certainembodiments, the methods and kits of the invention are useful fortreating complications arising from long-term hemodialysis regimens, forexample, hyperplasia, thrombosis, venous stenosis, arterial stenosis, ormorbidity. Such long-term complications typically develop as a resultsof multiple hemodialysis sessions, for example about 4, 6, 8, 10, 15,20, 25, 30, 35, 40 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, or 365 sessions.Hematoma or infection typically arise from the practice of hemodialysisin the short-term, for example, from a 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or 3 monthsof hemodialysis sessions.

In certain embodiments, the methods of the invention are utilized forabout one year, the percent morbidity is about 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, 10%, 9% or 8% in comparison to compression alonewithout the application of a coagulant or vasoconstrictor. In certainembodiments, the methods of the invention are utilized for about oneyear, the 5 year survival rate is increased to about 30%, 32%, 35%, 40%or greater in comparison to use of gauze alone. Utilizing the methods ofthe invention to reduce compression time, life is extended for about 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 2.5years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5years, or 10 years.

In certain embodiments, where the patient has a mature A-V fistula orA-V graft vascular access complications are reduced as a result of usingthe methods and kits of the invention and in comparison to the percentof complications that arise without the kits and methods of theinvention.

The percent of complications can be measured using imaging techniquesdescribed herein or compared to the known intervention rates or lengthof time until vascular access complications require a firstintervention, where intervention is needed due to vascular accesscomplications (Schwab, 1999, Kidney International, 55:2078-2090). Inaddition to reducing the number of vascular access complications, themethods of the invention reduce the severity of vascular accesscomplications. For example, the severity of stenosis may be determinedby the rate or amount of blood flow through the vascular access site orthe severity of thrombosis may be determined by measuring the frequencyand/or the dimensions of blood clots. Severity of hematomas may bedetermined by measuring the frequency and/or the dimensions of hematomasor the duration of a hematoma. In certain embodiments of the methods ofthe invention, the risk of short-term and/or long-term complications isreduced. In certain embodiments of the methods of the invention,reducing the risk means reducing the severity or number of short-termand/or long-term vascular access complications by about 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In certain embodiments, thereduced risk is in comparison to the same patient where the methods andkits of the invention are not used (e.g., using compression alone withgauze). In certain embodiments, the reduced risk is in comparison to thea patient population where the methods and kits of the invention are notused (e.g., using compression alone with gauze).

The compositions applied contribute to achieving a reduction orcessation of blood flow from the hemodialysis vascular access site, inparticular, the site of removal of hemodialysis needles, relative tocompression alone such that compression time can be shortened. Thisreduction in compression time in turn results in a reduction in vascularaccess complications associated with hemodialysis therapy, the averagenumber of vascular access sessions associated with a hemodialysistherapy, the infection rate associated with hemodialysis therapy, or apreservation of access to a hemodialysis vascular access site.

Generally, the methods of the invention, are intended for patients thatdo not normally, (e.g., over repeated hemodialysis sessions) exhibit acessation or reduction of blood flow out of, e.g., bleeding from, of thehemodialysis vascular access site in less than about one to aboutfourteen minutes, with compression in the absence of a composition ofthe invention. The methods and kits of the invention would provide thegreatest benefits for this class of patient. Some patients in thehemodialysis population have hemostasis times naturally fewer than about14 minutes. In certain embodiments, the methods of the invention are notutilized for patients who exhibit rapid hemostasis times usingcompression and gauze alone, for example hemostasis times of 9, 8, 7, 6,5 minutes or less.

4.2.1. Administration of Anticoagulants

In certain embodiments of the invention, the methods and kits of theinvention may be administered to a patient to whom an anticoagulant inan effective amount to prevent coagulation of blood has beenadministered. Examples of anticoagulants that may be used in conjunctionwith the invention include, coumadin™, dicumarol™, warfarin™, ecotrin™,heparin, nadroparin, aspirin, an antiplatelet drug, or a thrombolyticagent. Typically, full dose of heparinization for more than 12 hoursbefore a medical procedure is common. In other protocols heparin isadministered at repeated intervals to ensure that a constant activeclotting time is maintained (Falstrom et al., 1997, Catheterization andCardiovasular Diagnosis 41:79-84). In one embodiment, the anticoagulantcan be administered separately from the compositions of the invention,either concurrently or prior to administration of the compositions. Forthose patients to whom an anticoagulant has been administered prior tohemodialysis, the composition of the invention may further comprise oneor more agents that locally neutralize the effect of the administeredanticoagulant. In patients to whom heparin has been administered thecomposition may further comprise protamine sulfate in an amounteffective to locally neutralize heparin.

In certain embodiments of the invention, protamine sulfate in aneffective amount to locally neutralize heparin at the vascular accesssite is administered. In one embodiment, protamine sulfate can beadministered separately from the compositions of the invention, eitherconcurrently or prior to administration of the compositions.

4.2.2. Administration of Compositions Comprising a Vasoconstrictor

Following removal of the dialysis needles, a formulation comprising aneffective amount of a selected vasoconstrictor compound can be appliedto each needle site. The formulation can be directly applied to avascular access site as an adherent composition or may be embeddedwithin or coated upon another material that is held in place with anadhesive material. If necessary or desired, the formulation,composition, or material containing the composition is held in placewith firm digital pressure for a period of time that is less than about14 minutes, preferably, less than about 10 minutes, after which bleedingis arrested. In certain embodiments, the period of time is less thanabout 5 minutes.

For example, in the Examples presented herein below, the compositionwill adhere to the patient's skin at the vascular access site, or, canbe used to coat another material that is applied to the patient's skin.For example, the Examples presented herein below, the composition can becoated onto a barrier membrane to which is attached an oxygen-permeableTeflon backing. The backing extends beyond the edges of the barriermembrane and comprises an adhesive material suitable for holding thebarrier membrane to the patient's skin, thereby forming an adhesivepatch. For example, in the Examples presented herein below, the barriermembrane is a barrier-forming mat constructed withpoly-β-1→4-N-acetylglucosamine polymers and coated with a gel comprisingof the vasoconstrictor, endothelin-1. Following removal of the dialysisneedles, the Teflon-backed patch comprising endothelin-1-coated onto thepoly-β-1→4-N-acetylglucosamine-containing, barrier-forming mat isadhered to the vascular access sites. In the Examples presented hereinbelow, for example, digital pressure is not applied to the patch, whichremains attached to the patient's skin until removed by the patient athis/her convenience. However, in the Examples presented below, ifdigital pressure is applied, it is applied for a period of less than tenminutes, preferably less than about 5 minutes.

4.2.3. Administration of Compositions Comprising a Coagulant

Following removal of the dialysis needles, a formulation comprising aneffective amount of a selected coagulant can be applied to each needlesite. The formulation can be directly applied to a vascular access siteas an adherent composition or may be embedded within or coated uponanother material that is held in place with an adhesive material. Ifnecessary or desired, the formulation, composition, or materialcontaining the composition is held in place with firm digital pressurefor a period of time that is less than about 14 minutes, preferably,less than about 5 minutes, after which bleeding is arrested.

For example in the methods described in the Examples presented hereinbelow, the composition can be formulated such that it will adhere to thepatient's skin at the vascular access site, or, can be used to coatanother material that is applied to the patient's skin. In the Examplespresented herein below, for example, the composition is coated onto abarrier membrane to which is attached an oxygen-permeable Teflonbacking. The backing extends beyond the edges of the barrier membraneand comprises an adhesive material suitable for holding the barriermembrane to the patient's skin, thereby forming an adhesive patch. Inthe Examples presented herein below, for example, the barrier membraneis a barrier-forming mat constructed with poly-β-1→4-N-acetylglucosaminepolymers and coated with a gel comprising of the coagulant, tranexamicacid. Following removal of the dialysis needles, the Teflon-backed patchcomprising tranexamic acid-coated onto thepoly-β-1→4-N-acetylglucosamine-containing, barrier-forming mat can beadhered to the vascular access sites. For example, in the Examplespresented herein below, digital pressure is not applied to the patch,which remains attached to the patient's skin until removed by thepatient at his/her convenience. However, if digital pressure is applied,it is applied for a period of less than ten minutes, preferably lessthan about 5 minutes.

4.2.4. Applying Compression

Compression can be applied to the vascular access site in several ways.The present methods can entail a combination of applying pressure andcontacting a barrier-forming material and/or a composition comprising avasoconstrictor and/or coagulant agent to a site on the patient's skinsurface where hemodialysis needles have been removed that is contiguouswith the vascular access site. Typically, the site of application is thewound where the hemodialysis needles have been removed.

In certain embodiments of the invention, the compression is manualcompression. Manual compression can, for example, be applied by pressingwith the tips of fingers on the skin surface, at a point above anunderlying vascular access site, so that the vascular access site iscompressed and blood flow is significantly reduced or stopped in thetargeted vascular access site. This type of compression can be appliedat a site proximal of removal of hemodialysis needles in a vascularaccess site. Typically, a proximal site is between about 1 and about 10cm proximal of the removal site. In certain embodiments of theinvention, manual compression is applied directly to the site where thecomposition has been applied to the vascular access site. Typically thecomposition is applied topically to the portion of the vascular accesssite that is bleeding. The technique of manually pressing on a patient'sskin at the correct place to reduce blood flow and blood pressure iscommon in the art of and is effective in both humans and other mammals(Camenzind et al., 1994, Journal of the American College of Cardiology.24(3):655-662; Kipshidze et al., 1998, Journal of Invasive Cardiology.10(3):133-141; Merino et al., 1992, Catheterization and CardiovasularDiagnosis 26:319-322; Sanborn et al., 1993, Journal of the AmericanCollege of Cardiology. 22(5):1273-1279).

The manual compression can be applied in combination with application ofa composition comprising a vasoconstrictor and/or coagulant. Suchembodiments can entail manually applying compression to a vascularaccess site concurrently with applying a composition as described hereinabove. Alternatively, manual compression can be applied immediatelyprior to application of a composition or immediately after applicationof a composition.

In an embodiment of the invention that encompasses a manual compressiontechnique as a part of a method of the invention, the technique may beone wherein pressure is applied with one's fingers or the palm of one'shand directly over a vascular access site. In certain embodiments,compression can be applied directly to the wound site at the same angleat which the needle was inserted. In one embodiment of the invention,manual compression may be applied concurrently with application of thecomposition. If the composition of the invention is formulated as abarrier-forming material, then the composition and the compression canbe applied at the same time. If the composition is, for example, a gel,the gel can be applied to the hemodialysis needle removal site followedimmediately by compression, or the gel might be applied to abarrier-forming material such as gauze and the treated gauze can then beapplied at the same time as compression.

In applying manual compression techniques, the amount of pressureapplied and the consistency of pressure force generally varies over timeand among individuals applying the pressure. This is satisfactory aslong as post-hemodialysis bleeding is reduced or ceases in about one tofourteen minutes, such that compression can be removed in about one toabout fourteen minutes.

In certain embodiments, it may be preferable to apply compression to avascular access site mechanically rather than manually. Several deviceswell known to those of skill in the art have been developed and arecommonly used to apply compression, including C-clamps of varying oradjustable force, and compression bandages/dressings coupled withattachment of desired weights. Such compression devices may be used inconjunction with the methods of the invention and can provide a constantapplication of the desired compression force. In a preferred embodiment,the amount of compression force applied to the vascular access siteallows for blood flow through the vascular access site. In a relatedembodiment, the blood flow is about 50, 10, 200, 300, 400, or 500ml/min. In a related embodiment, the blood flow is about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% of the normal physiological flow rate through the vascularaccess site.

If non-manual compression is employed in the methods of the invention,then a constant pressure may be applied. A technique of using stepwisereduction in compression can be employed when using for example aC-clamp or compression bandage/dressing, wherein the pressure may belessened in increments over time as necessary. A fluid-filled balloonconnected to a pressure transducer can also be used to measure theamount of pressure applied.

In certain embodiments, compression is either applied first at a siteproximal to the vascular access site to decrease blood flow in thevascular access site or is applied first to both the proximal site andthe vascular access site simultaneously; in either case the compressionapplied at the proximal site is later released or decreased while thecompression at the vascular access site is maintained. The time betweenthe application of compression and the release of pressure at theproximal site can be about 15 seconds, 30 seconds, 1 minute, 1.5minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5minutes, or 5 minutes. In other embodiments, compression is applied andmaintained at both the proximal and vascular access sites.

The amount of compression force varies for each patient and vascularaccess site being treated. Any of the compression techniques describedherein can be used in carrying out the methods of the invention, as wellas other standard compression techniques. Various methods for measuringcompression time and compression force can be employed in carrying outthe methods of the invention as described above.

4.2.5. Compression Time

One of the advantages of the methods of the present invention is thatthe combination of compression and the compositions results in areduction in compression time needed to reduce or cease the flow ofblood from the vascular access site which leads to a reduction invascular access complications relative to patients who receive longerperiods of compression. The amount of time for which compression ismaintained to achieve hemostasis, i.e., a cessation or reduction ofblood flow from a skin surface wound contiguous with a vascular accesssite, is subject to the size of the wound and the period of time forwhich pressure is applied in compressing the blood vessel. As usedherein in connection with the invention, hemostasis means cessation orreduction of blood flow from a skin surface wound contiguous with avascular access site. The amount of time for which compression ismaintained when carrying out the methods of the invention is shorter forthe majority of hemodialysis sessions in comparison to compression aloneor compression without a composition as measured under comparablecircumstances.

In one embodiment of the invention, the compression time necessary tocease or reduce bleeding is less time than necessary when applyingcompression in conjunction with a topical barrier-forming materialwithout a vascoconstrictor and/or coagulant. In another embodiment, thecompression time is about one to fourteen minutes, for the majority ofhemodialysis sessions in comparison to applying compression inconjunction with a topical barrier-forming material without avasoconstrictor and/or coagulant, and vascular access complicationoccurrence decreases.

In certain instances, the period of time for which compression isapplied may be fewer than about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5,7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14minutes. In certain embodiments, the period of time for whichcompression is applied is greater than 11 minutes. In certainembodiments, the period of time for which compression is applied isabout 11 to 25 minutes. In one embodiment, compression is applied for aperiod of about one to fourteen minutes, for the majority ofhemodialysis sessions. The period of time begins after the hemodialysisneedles are removed at the time compression is initially applied. Theperiod of time is continuous until compression is released for the finaltime during the session. The period of time can include intervals oftime during which compression is released momentarily to check forbleeding from the wound. In one embodiment, the period of time caninclude only the periods of time during which compression is applied,excluding the breaks to observe or measure bleeding.

In certain embodiments of the methods and kits of the invention wherethe composition comprises a vasoconstrictor and for a coagulant, thecompression time is reduced by about 2%, 5%, 8%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% in comparisonto compression in conjunction with a barrier-forming material having novasoconstrictor and/or coagulant.

The time for applying compression, manual or otherwise, in conjunctionwith a composition of the invention may be divided into intervalsbetween which there is a release or partial release of compression, forexample, in order to record observations to calculate the cessation rateor time of blood from a wound and/or to calculate the time to achievehemostasis. The compression intervals may, for example, be about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds in length.Alternatively, the time intervals may be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 minutes in length. In apreferred embodiment, the bleeding is checked once about half waythrough the desired time of compression. In a related embodiment, thebleeding is checked after about 5 minutes of compression. Generally, theperiods between compression intervals are about 1-10 seconds in length.

In another embodiment, compression is applied for time intervals ofequivalent length. The total compression time continuous or interruptedto determine bleeding time is about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, or 14.5 minutes. In one embodiment, compression is appliedto the desired vascular access site immediately following the removal ofthe hemodialysis needles.

The compositions described in section 4.1. are preferably appliedconcurrently with an initial application of compression in the methodsof the invention. In other embodiments, the compositions can bere-applied at compression intervals. In other embodiments, compressionis applied for a time period prior to application of a composition andfurther compression. In one embodiment, the time period prior toapplication of a composition is about 1, 2, 3, 4, or 5 minutes.

4.2.6. Measurement of Time or Rate of Blood Flow or Cessation ofBleeding

In certain embodiments, the rate or time of cessation or reduction ismeasured to determine if cessation or reduction of blood flow (e.g.,bleeding) out of a hemodialysis needle wound is achieved in about one toabout fourteen minutes. The rate or time of cessation or reduction inthe presence or absence of a composition can also be measured forcomparison purposes.

Additional techniques that can be used with the methods of the inventiondescribed herein to observe cessation or reduction of blood flow out ofthe vascular access site or rate of blood flow through the vascularaccess site are taught below.4.2.5.

The rate of or time to achieve cessation or reduction of blood flow outof the vascular access site can be calculated simply as the total numberof time intervals required to achieve the effect, provided the timeintervals are equivalent for each treatment, i.e. compression alone orcompression in conjunction with the composition of the invention. Forexample if the sum of the number of time intervals required for allpatients receiving a first treatment is 8 and if the sum of number oftime intervals required for all patients receiving a second treatment is10, then the percent difference would be 25%. Alternatively, the averagetime to achieve cessation or compression time could be calculated foreach treatment group, then compared to determine the percent difference.

In another embodiment, the time to achieve cessation of compression timeis measured in minutes and/or seconds rather than time intervals. Thepercent difference between the times measured can be calculated with oneof the methods described above for the time intervals method.

In certain embodiments of the invention, the hemodialysis patient has amean hemostasis time of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30minutes. In certain embodiments, the patient has a mean hemostasis timegreater than 11 minutes.

Where the time to achieve cessation or reduction of blood flow from thevascular access site caused by removal of hemodialysis needles from avascular access site is measured in minutes and/or seconds, the numberof time intervals for which compression is applied may not necessarilybe a factor in calculating the time to cessation. Total time fromapplication of treatment to cessation or reduction of blood flow can becompared for the two treatment groups. In this embodiment of theinvention, compression alone may be initially applied for a longerperiod of time than compression in conjunction with a composition asdescribed herein above in section 4.2.5. since values for the time toachieve cessation are known in the art for compression alone. The timeto achieve cessation or the rate of cessation of blood flow can also bemeasured for the circumstance where only compression is applied. Theabove described measurements can be made by imaging methods known to oneof skill in the art, including but not limited to ultrasonography andscintigraphic imaging. For example, compression alone may be applied toa patient for five minutes before an observation is recorded, while thepatient receiving compression in conjunction with the composition of theinvention would require compression to be partially and briefly releasedto make blood flow observations, in order to obtain data to calculatethe time to achieve cessation or total compression time for ahemodialysis session.

If a set number of compression time intervals is applied, the percentdifference can be calculated based on the number of patients in eachtreatment group. For example, if compression is applied for two timeintervals and 60 of 100 patients receiving a first treatment consistingof compression alone achieve cessation of bleeding after two compressionintervals, and 80 of 100 patients receiving a second treatmentconsisting of compression plus the composition of the invention achievecessation or sealing of a wound after two equivalent time intervals,then the percent difference in time to achieve hemostasis or the totalcompression time for a hemodialysis session would be 20%.

The frequency of thrombosis occurrence or other vascular assesscomplications can be calculated with clinical observations. Oneindicator of thrombosis is reduction of blood flow through the vascularaccess site. The reduction of blood flow through the vascular accesssite can be in comparison to the flow rate before the hemodialysissession started or physiological standard flow rate which could bereadily determined from standard references. Alternatively, diagnoses ofstenosis or other vascular complications as well as compression time toachieve cessation of blood flow and amount of sealing of the wound mayalso be made with various imaging techniques.

The reduction in vascular access complications can be measured orquantified in several ways. For example, comparisons can be made amonggroups of patients, where one group serves as a control and receivesstandard hemodialysis therapy and another group receives hemodialysistherapy with the methods of the invention. The mean numbers of vascularaccess complications can then be compared for the groups to identifyreductions in the occurrence of vascular assess complications. Thereduction in vascular access complications can also be measured within apatient by comparing the numbers of vascular access complications duringperiods of hemodialysis sessions with and without the methods of theinvention. Standard medical statistical methods can be used to identifyreductions, such as those methods described below.

In various embodiments, vascular access complications decrease by about5%, 10%, 20%, 30%, 40, 50%, 60%, 70%, 80%, 90% or 100% for a patient orpatient population administered the methods of the invention.

In one aspect of the invention, the rate of cessation or reduction ofblood flow when employing the methods of the invention is 10% greaterthan applying compression in conjunction with a topical barrier or gauzewithout a vasoconstrictor and/or coagulant. In other aspects, the rateas described above is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,70%, 80% or greater.

In one aspect of the invention, the time to achieve cessation orreduction of blood flow when employing the methods and compositions ofthe invention is 10% less than applying compression in conjunction witha topical barrier or gauze without a vasoconstrictor and/or coagulant.In other aspects, the time as described above is 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% or less relative to applyingcompression alone (e.g., gauze alone).

Statistical methodology can be employed to determine if the observeddifferences are statistically significant. In particular, thedifferences observed between the compression time or occurrence ofvascular complications using the methods of the invention in comparisonto compression alone or compression with vasoconstrictors and/orcoagulants can be analyzed with standard statistical methodology.Statistical significance can be determined with any standard calculatedstatistic (e.g., a one-tailed t statistic, a two-tailed t statistic, achi square statistic, an F statistic, etc.). Standard statisticalmethodologies suitable for use in connection with the invention includemethodologies commonly used in medical analyses and clinical trials.Examples of methodologies can be found in reference publicationsincluding, but not limited to: Vogt W., 1998, Dictionary of Statisticsand Methodology 2nd ed., SAGE Publications; Spiegel, D., J. Myles, andK. R., 2002, Abrams Bayesian Approaches to Clinical Trials and HealthCare: Statistics in Practice. Wiley, John & Sons, Incorporated;Cleophas, T. J., A. H. Zwinderman, and T. F. Cleophas, 2002, StatisticsApplied to Clinical Trials 2nd ed., Gehan, E. A. A. and N. A. Lemak,1994, Kluwer Academic Publishers; and Statistics in Medical Research:Developments in Clinical Trials 1st ed., Kluwer Academic Publishers.

For example, color flow duplex sonography of a vascular access site canbe used to test for pseudoaneurysms (Gwechenberger et al., 1997,Angiology. 48(2):121-126.) Scintigraphic Image Analysis can be employedto examine blood flow and/or clott formation at the site of removal ofhemodialysis needles or the site of A-V connection or graft and todetermine if a wound in a vascular access site caused by a hemodialysisneedle is effectively sealed (Ismail et al., 1995, Catheterization andCardiovasular Diagnosis 34(1):88-95). Angiogram technology can also beused to examine sealing of a wound site caused by hemodialysis needles(Hoekstra et al., 1998, Biomaterials. 19:1467-1471). hyperplasia can bemonitored and diagnosed as a vascular access complication using, forexample, intravascular ultrasound (Schwab, 1999, Kidney International,55:2078-2090). With imaging analyses as described herein, the time rateand percent difference in rates or times of cessation or reduction ofblood flow from the hemodialysis needle wound or blood flow through theA-V fistulae or graft can be calculated without releasing compression tomake observations. The number of vascular access complications, e.g.thrombosis events, can also be calculated with the imaging techniquesdescribed herein or with other imaging techniques well known to those ofskill in the art of diagnosis vascular access complications.

In one embodiment, the methods of the invention comprise the step ofmeasuring or monitoring the presence or development of vascular accesscomplications or blood flow through the vascular access site. This canbe achieved using, for example, the imaging techniques described below.Such monitoring may be done at each hemodialysis session or at fixedperiods of time, such as every 3 months. Alternatively, the monitoringcan be done if the patient exhibits a history of symptoms of vascularaccess complications or clinical manifestation of vascular accesscomplications.

The blood flow rate measured can be during compression, prior tocompression, subsequent to compression, or blood flow rate thought thehemodialysis machine. In certain embodiments, the blood flow through thevascular access site is measured in comparison to blood flow through thesite prior to compression, at maturity of the site prior to repeateduse, or physiological flow rate through the site. Schwab et al. (1989,Kidney International, 36:707-711) proposed detection of venousanastomosis, an indicator of access site thrombosis, where detection wasachieved by monitoring the venous pressure at a set blood flow rate of200 ml/min during the dialysis procedure. Other techniques formonitoring or measuring blood flow through a vascular access siteinclude recirculation (i.e., when blood pump flow demand fromhemodialysis machine exceeds access blood flow as a result of latevenous outlet stenosis), acute drop in dose of dialysis, access bloodflow (i.e., the lower the blood flow velocity, the higher the risk ofthrombosis: blood flows of ≧800 ml/min have a higher rate of thrombosisthan blood flows of ≧1,000 ml/min in PTFE grafts and in A-V fistulaevascular access complication risk rises if blood flow is less than about400 ml/min, or in either type of access a decrease in blood flow of 25%is indicative of vascular access complication risk), and ultrasounddilution, angiography, Intravenous digital subtraction angiography(DSA), Doppler Ultrasound, or duplex doppler color flow for measuringblood flow velocity and observing anatomical pathology.

In embodiments of the invention, where blood flow through the vascularaccess site is measured to determine the reduction in vascular accesscomplications or to extend the time a vascular access site can be usedfor hemodialysis, blood flow is greater than about 600 ml/min, 650ml/min, 700 ml/min, 750 ml/min, 800 ml/min, 850 ml/min, 900 ml/min, 950ml/min, or 1000 ml/min in the vascular access site when the methods andkits of the invention are employed and blood flow is measured prior toor following a hemodialysis session. In related embodiments, the bloodflow rate in the vascular access site is less than 1500 ml/min.

In a preferred embodiment, the methods of the invention effectivelyreduce the likelihood of vascular access complications, includingthrombosis.

In certain embodiments of the methods and kits of the invention wherethe composition comprises a vasoconstrictor and/or a coagulant, thenumber, rate, or percent of patients with vascular access complicationsis reduced by about 2%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more in comparisonto compression in conjunction with a barrier-forming material having novasoconstrictor and/or coagulant, where compression is applied for aboutthe same period of time.

In certain embodiments of the methods and kits of the invention wherethe composition comprises a barrier-forming material not having avasoconstrictor and/or a coagulant, the number, rate, or percent ofvascular access complications is reduced by about 2%, 5%, 8%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or more in comparison to compression for the clinical standardtime, e.g., 15-20 minutes, in conjunction with a barrier-formingmaterial, e.g., gauze, having no vasoconstrictor and/or coagulant.

4.2.6.1. Scintigraphic Image Analysis

Scintigraphic Image Analysis can be employed to examine blood flowand/or clot formation at site of removal of hemodialysis needles or avascular access site and to determine if a wound is effectively sealed,especially in animal model systems (Ismail et al., 1995, Catheterizationand Cardiovasular Diagnosis 34(1):88-95). For example, if the inventionis being tested in an animal model, a camera fitted with a low energyall-purpose collimator can be placed in position over a hemodialysisvascular access site. Point sources (˜50 uCi 99 mTc) in suitablecontainers may be used to record the exact position of the animal body,for alignment of subsequent images. The test animals are injectedthrough with autologous technetium-labeled red blood cells,(mean=16.65±3.51 mCi), for which the labeling efficiency is previouslytested. The labeled cells are allowed to circulate for 5 minutes beforeimages are taken. Images can be taken at regular time intervals up to 24hours. Early images can be used to ascertain sealing of a hemodialysiswound and cessation or amount of blood flow, whereas later images can beused to identify clotts with great efficiency.

4.2.6.2. Angiography

An angiogram is a test in which a dye, or contrast, is administered to apatient and X-ray imaging is used to look inside blood vessels in orderto diagnose or treat lesions involving the blood vessels. Angiogramtechnology is commonly employed to guide catheters and needles.Angiograms can also be used to examine, in a non-invasive manner,cessation or rate of blood flow, or sealing of such a breach or puncture(Hoekstra et al., 1998, Biomaterials. 19:1467-1471). CT or MRI imagingcan also be used to examine sealing of a hemodialysis needle wound siteand cessation of blood from the wound, blood flow through the vascularaccess site, stenosis, or development of vascular access complications.

4.2.63. Ultrasonography

Various sonography and ultrasonography techniques may be employed withthe methods of the invention to examine bleeding and/or clotting at thesite of removal of hemodialysis needles or bleeding and/or presence of avascular access complication at a vascular access site. Duplexultrasonography has demonstrated useful clinical applications inperipheral arterial testing for lesion localization and quantificationof abnormal blood flow. For example, color flow duplex sonography of apuncture wound site can be used to test for blood clotts,pseudoaneurysm, and A-V fistula formation (Gwechenberger et al., 1997,Angiology. 48(2):121-126).

4.3. Composition Dosage and Administration

Generally, a therapeutically effective amount, will vary with thepatients age, condition, and sex, as well as the nature and extent ofthe condition in the subject, all of which can be determined by one ofordinary skill in the art. A therapeutically effective amount is anamount of composition that achieves hemostasis in about one to aboutfourteen minutes, with compression. For example, the effective dose(i.e., amount) needed for an infant may differ from an elderly patient.The actual amount and formulation of the composition to be administeredwill depend on various factors such as the severity of the wound, thecondition of the patient, the age of the patient and any collateralinjuries or medical ailments possessed by the patient. For example, ESRDpatients often have cardiac disease, compromised vascular systems,hypertension, hypotension, diabetes mellitus, polycystic kidney disease,glomeralonephritis or infections diseases, e.g., HIV or hepatitis.

Toxicity and efficacy of the compositions of the invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the ED50 (the dosetherapeutically effective in 50% of the population). Compositions thatexhibit greater therapeutic effect are preferred. In the presentinstance, compositions that exhibit toxic side effects may be used incarrying out the methods of the invention. The potential damage tounaffected cells is minimized, since the compositions are applied to thesite of affected tissue and thereby reduce the risk of side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compositions lies preferably within a range of concentrations thatinclude the ED50. The dosage may vary within this range depending uponthe formulation of the composition, i.e., gel, foam, patch, etc. For anyvasoconstrictor used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from tissue or tissue cultureassays.

One standard tissue assay is conducted using aortic rings excised fromrats. The aorta are then rapidly suspended in a warmed Krebs-Henseleit(KH) buffer consisting of (in mmol/l): 118 NaCl, 4.75 KCl, 2.54CaCl₂.2H₂O, 1.19 KH₂PO₄, 1.19MgSO₄.7H₂O, 12.5 NaHCO₃, and 10.0 glucose.Isolated vessels can be carefully freed of connective tissue and cutinto rings 2-3 mm in length. The rings are then mounted on stainlesssteel hooks, suspended in a 10-ml tissue bath, and connected to FT-03force displacement transducers (Grass Instrument, Quincy, Mass.) torecord changes in force on a Grass model 7 oscillographic recorder. Thebaths are filled with KH buffer and aerated at 37° C. with 95% O₂+5%CO₂. A resting force of 0.5 g is applied to the SMA rings, and then therings are equilibrated for 90 minutes. During this period, the buffer inthe tissue bath is replaced every 15-20 minutes, and the resting forceof the vascular rings is adjusted until 0.5 g of pre-load is maintained.After 90 to 120 minutes of equilibration, the rings are exposed to 100nM U-46619 (9,11-dideoxy-9α-11α-methaneepoxy-prostagalandin F_(2 α),Biomol Research Laboratories, Plymouth Meeting, PA), a thromboxane A₂mimetic, to generate 1.0 g of developed force. Once a stable contractionis obtained, acetylcholine, a typical endothelium-dependent vasodilator,is added to the bath in cumulative concentrations of 0.1, 1, 10, and 100nM to assess the integrity of endothelium. After the cumulative responseis stabilized, the rings are washed and again allowed to equilibrate tobaseline. Once a stabilized baseline response is obtained one skilled inthe art can proceed to test various vasoconstrictors by repeating theprocedure.

The function of a coagulant can be tested by standard assays. In suchassays, normal human blood, without anticoagulant, is drawn and placedin several test tubes. For Example, the normal blood, without acomposition of the invention, is allowed to clot (usually within about10 minutes). Other samples of normal blood are drawn and one milliliteraliquots are placed in test tubes with descending aliquots of aparticular composition of the invention for which one desires to testcoagulant properties. Variations on this standard assay can be conductedwhere the patient has had an anticoagulant introduced into thebloodstream prior to withdrawal of blood. The results can be used toidentify compositions of the invention that can accelerate hemostasis,i.e. cessation of flow of blood from a wound.

In various embodiments, an amount of vasoconstrictor and/or coagulanttested for effectiveness is an amount that is about 0.5-fold, 0.75-fold,1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 12-fold, 15-fold,20-fold, 50-fold or 100-fold of effective dosage that can be used in thepresent methods.

In certain embodiments of the invention, an effective amount of avasoconstrictor and/or coagulant is an amount that results in reductionor cessation of bleeding when applied to a catheter exits wound withoutcompression. In other embodiments of the invention, an effective amountof a vasoconstrictor and/or coagulant is an amount that results inreduction or cessation of bleeding when applied to a catheter exitswound with compression according to the methods of the invention.

In embodiments of the invention where the composition of the inventionis formulated as, embedded in, or applied to a patch, 100 mg of thecomposition may be present in 1 cm² of the wound-contacting surface ofthe patch. In other embodiments, the effective amount of a compositionof the invention present in 1 cm² of a patch can be about 0.05 mg, 0.10mg, 0.25 mg, 0.50 mg, 0.75 mg, 1 mg, 2 mg, 5 mg, 8 mg, 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg,350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg,800 mg, 850 mg, 900 mg, 1000 mg, or 2000 mg of the composition, whereinthe composition has a concentration between about 1 mM and 70 mM. In apreferred embodiment, wherein the composition comprises p-GlcNAc, theeffective amount of a p-GlcNAc present in 1 cm² of a patch is between 3mg and 20 mg of the composition.

One skilled art would readily be able to determine the amount of avasoconstrictor and/or a coagulant effective to achieve hemostasis andreduce the need for compression. For example, standard doses and methodsfor determining dosage of drugs are described in the Physicians' DeskReference (Medical Economics Company, Inc., Montvale, N.J., 2000,) whichis incorporated by reference herein in its entirety. The methodsdescribed in section 4.2.5. for calculating hemostasis time by observingblood flow at time intervals can also be used to determine whether aspecific amount of a specific vasoconstrictor and/or a specificcoagulant is an effective amount such that one applying compression cando so for about one to fourteen minutes, and achieve hemostasis.

In other embodiments, where the composition of the invention isformulated as, embedded in, or applied to a patch, 100 μg of thecomposition may be present in 1 cm² of the wound-contacting surface ofthe patch. In other embodiments, the effective amount of a compositionof the invention present in 1 cm² of a patch can be about 5 μg, 10 μg,20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 105 μg, 110 μg, 115 μg,120 μg, 125 μg, 130 μg, 135 μg, 140 μg, 145 μg, 150 μg, 155 μg, or 160μg of the composition, wherein the composition has a concentrationbetween about 1 mM and 70 mM. An example of a coagulant that can beeffective in such amounts is thrombin. An example of a vasoconstrictorthat can be effective in such amounts is endothelin-1.

In yet other embodiments, the effective amount of a composition of theinvention is about 1-1000 IU/cm² wherein the composition is formulatedas, embedded in, or applied to a patch.

In certain embodiments, an effective amount of a composition of theinvention is an amount that activates hemostasis in the presence of acoagulant or an anticoagulant.

In certain embodiments, the effective dose is the dose necessary toinitiate clotting with or without compression. In other embodiments, theeffective dose is the dose necessary to cause formation of a firm clotthat will remain with or without compression. In yet other embodiments,an effective dose can be determined by the strength of the clot, i.e.the time for which the clot holds with or without compression.

Once it has been determined how varying concentrations and amounts of aparticular vasoconstrictor and/or coagulant act in vitro, effectivevasoconstrictors and/or coagulants can be further tested in animalmodels by methods. A series of measurements can be taken varying theconcentration or amount of the vasoconstrictor and/or coagulant todetermine an effective amount. Such series of measurements can be usedto predict how a particular vasoconstrictor and/or coagulant willfunction and allow for a determination of effective amount for apatient. Such information can be used to more accurately determineuseful doses in humans.

Results from animal models can be extrapolated to determine effectivedoses for human subjects. Comparing varying concentrations of avasoconstrictor and/or coagulant in one or more animal models allows forthe establishment of dose response curves that can be used to estimateeffective amounts in a human, given the particular circumstances of eachsubject, i.e., size of wound, age of patient, presence of coagulants oranticoagulants in the blood stream.

Ultrasound imaging or Doppler flow analysis of the vascular access siteallows for the determination of cessation or reduction of blood flowthrough the skin surface wound or vascular access site. If blood flowthrough the vascular access site is inhibited for prolonged periods oftime, i.e., greater than about fourteen minutes, it may cause vascularaccess complications. Thus, Doppler flow and ultrasound would allow fora determination of the maximum upper limit of an effective amount of avasoconstrictor and/or coagulant. For example, if the effect of acoagulant extends into the blood vessel and causes clotting of plateletand cessation of blood within the blood vessel, the effect could bedamaging. In another embodiment, the maximum upper limit of an effectiveamount of a vasoconstrictor and/or coagulant can be measured as theamount of the composition of the invention that causes a cessation ofblood flow through the vascular access site in the presence oralbescence of compression for about one to fourteen minutes.

4.4. KITS

A kit is also provided which according to the invention comprises any ofthe above described embodiments. The kit can include the compositioncontained within a sealed, water proof, sterile package whichfacilitates removal of the composition without contamination. Materialsfrom which containers may be made include aluminum foil, plastic, oranother conventional material that is easily sterilized. The kit cancontain a single composition or multiple compositions, preferablywherein each is provided in a separate, waterproof, sterile package. Inone embodiment, the kit can include the composition contained within aplastic or metal tray or container with one or more compartments thatprovides a barrier to moisture.

In another embodiment, a container having dual compartments is provided.A first compartment contains the composition, while the secondcompartment contains a topical barrier, membrane, or film according tothe invention. In field use, the barrier, membrane, or film can bereadily dipped into an opened first compartment and subsequently appliedto the hemodialysis needle wound. The composition can be applied oradded to a topical barrier, membrane, or film prior to being packagedand sterilized or the composition can be formulated as a barrier,membrane, or film. In another embodiment, the kit may not contain abarrier, membrane, or film if the composition is not formulated as such.

According to one aspect of the invention, various specialized kits canbe provided. The kit can contain multiple compositions of the invention,wherein each is contained within a separate sealed sterile package orcontainer. The kit can contain in one or more containers, a an effectiveamount of a vasoconstrictor, while in another separate container apharmaceutically acceptable carrier can be packaged. In a separatecontainer the kit can contain a neutral liquid, neutral gel or neutralsolid. The kit may also contain a coagulant in accord with thecomposition for use in the methods of the invention as described herein.

The kit can comprise a composition that can be formulated as abarrier-forming material that forms a barrier to blood. The kit cancomprise a composition that can coat, be added to, or integrated into abarrier-forming material that forms a barrier to blood. In oneembodiment a kit comprises a pharmaceutical composition comprising apatch made of barrier-forming materials that are embedded with one ormore coagulant and/or vasoconstrictor agent(s). In one embodiment a kitcomprises a pharmaceutical composition comprising a gauze embedded withone or more coagulant and/or vasoconstrictor agent(s). In certainembodiments, a kit comprises pharmaceutical compositions comprising abarrier-forming material embedded or combined with one or more coagulantand/or vasoconstrictor agent(s), wherein the barrier-forming materialcontains an adhesive so that the material can be adhere to a patient'sskin surface. Alternatively, a kit lacks barrier-forming materials.

In one embodiment a kit comprises a patch embedded or coated with avasoconstrictor, a coagulant, or an agent that functions as both avasoconstrictor or coagulant. In another embodiment a kit comprises agel that can be used in the methods of the invention, wherein the gelcomprises a vasoconstrictor, a coagulant, or an agent that functions asboth a vasoconstrictor or coagulant.

In a preferred embodiment the kit contains gauze.

A kit can comprise a notice regarding FDA approval and/or instructionsfor use in combination with compression during hemodialysis sessions.The instructions can recite a method for reducing vascular accesscomplications. In one embodiment, the instructions recite applyingtopically to a hemodialysis vascular access site during a hemodialysissession a composition comprising an amount of a vasoconstrictor orcoagulant effective for reducing post-hemodialysis bleeding at thevascular access site, and applying compression to the hemodialysisvascular access site for a period of about one to fourteen minutes. Theinstructions can also recite post procedural instructions, warnings,precautions, and/or indications of market clearance or approval. Theinstructions can also recite instructions for labeling the kit and/orkit components.

In one embodiment, the kit comprises instructions for topically applyingthe composition at a hemodialysis vascular access site where compressionis applied for about one to fourteen minutes, resulting in a reductionin vascular access complications.

In another embodiment, the kit comprises instructions for topicallyapplying the composition with compression at a hemodialysis vascularaccess site to achieve a cessation or reduction of blood flow out of thehemodialysis vascular access site in about one to fourteen minutes.

5. EXAMPLES 5.1. Example 1 Use of the HealTekPatch™ to Promote RapidWound Closure and Cessation of Bleeding in Hemodialysis Patients

Example 1 is directed toward a study testing the utility of a particularmaterial, HealTekPatch™, as a device to promote rapid wound closure andcessation of bleeding from such vascular access sites in hemodialysispatients following needle removal. HealTekPatch™ fully-acetylatedpoly-N-acetyl glucosamine (“p-GlcNAc”). The polymer p-GlcNAc andderivatives and formulations thereof has been described in

U.S. Pat. Nos. 5,622,834; 5,623,064; 5,846,952; 5,624,679; 5,858,350;5,635,493; 5,686,115; 6,063,911; and 5,510,102.

The focus of the initial test of this example, was directed toward arandomly-selected group of 25 hemodialysis patients from within a totalpool of 66 patients who had met the inclusion criteria and who hadsigned a consent form. In this study, each patient served as his/her owncontrol.

As indicated by the data provided below, HealTekPatch™ proved moreeffective than the gauze control in 86% of the patients tested. Inaddition, use of HealTekPatch™ stopped bleeding within 5 minutes in 72%(18 of 25) of patients. Standard care (gauze) stopped bleeding within 5minutes in only 16% (4 of 25) of patients. Furthermore, within thesubset of patients treated with prophylactic anticoagulants, use ofHealTekPatch™ was more effective than the gauze control for rapid woundclosure and cessation of bleeding in 88% of the patients.

5.1.1. Materials

HealTekPatch™ (Marine Polymer Technologies, Danvers, Mass.) afully-acetylated poly-N-acetyl glucosamine (p-GlcNAc), is a highlypurified polysaccharide free of protein and other contaminants, seeSection 4.1.4. Biological testing of the HealTekPatch™ was carried outat Toxikon Laboratory (Woburn, Mass.), and included the following tests:Cytotoxicity, Primary Skin Irritation, Sensitization Assay, SystemicToxicity, Hemocompatibility, Pyrogenicity, Implantation Test,Mutagenicity, and Subchronic Toxicity. HealTekPatch fully biocompatibleand non-toxic, as indicated by United States Food and DrugAdministration (FDA) Tripartite Biocompatibility Guidance Tests. Thegauze used was Medical 2″×2″ 4 ply (Johnson & Johnson, New Brunswick,N.J.). The gauze was folded to form a 1″×1″ (2.5 cm×2.5 cm) square.

5.1.2. Patient Selection Method

Twenty-five patients were randomly selected for inclusion in the presentstudy from among the 66 hemodialysis-patient population on record at agiven date at Dialysis Clinic Inc. (Boston, Mass.).

Patients were offered the opportunity to participate in the study whenthey appeared for their routine hemodialysis treatments. A patient wasdeemed eligible for the present study if he or she: (1) was over 18years of age and able to give consent, (2) was dialyzed via native veinfistula or via polytetrafluoroethylene graft, and (3) had bled for morethan 5 minutes after removal of hemodialysis needles in a prior,observational study. Patients with antibodies to HIV or with Hepatitis Bsurface antigen were excluded from the study. Also excluded werepatients enrolled in other clinical trials. Table 1 summarizes thedemographics of the twenty five patients enrolled in the present study.

TABLE 1 Sex Female 11 44% Male 14 56% Age Under 60 6 24% 60-69 7 28%70-79 5 20% Over 80 7 28% Race Asian 6 24% Black 7 28% Hispanic 1 4%White 11 44% Vascular Access Fistula 15 60% Graft 10 40% ConcurrentDiseases Diabetes 6 24% Hypertension 6 24% None 13 52% Concurrent DailyProphylactic Anticoagulation Therapy aspirin 6 24% Ecotrin 3 12%Warfarin 4 16% None 12 48%

Patient evaluation included a review of the patient's clinical historyand medical chart as well as an inspection of the vascular access site,in order to ensure that the patients chosen met study inclusioncriteria. This patient evaluation also provided relevant clinical datafor each patient, which included: skin condition, the pattern of needlepunctures, the type of vascular access, heparin doses, venous pressures,blood flows, adequacy of dialysis, nutritional status, hematologicparameters, use of oral anticoagulation or antiplatelet therapy and ahistory of repairs to and replacements of the vascular access. Theresults of each patient's blood tests, which included the most recentcomplete blood count and platelet count, were copied from each patient'smedical chart.

5.13. Treatment Method

Each patient served as his/her own control. Each patient was treatedonce with the HealTekPatch™, and once with a gauze control (standardtreatment) to determine which method stopped bleeding faster. The orderin which each material (HealTekPatch™ disk or gauze square) was used,was randomized for each patient. Following removal of the dialysisneedles, the selected material (either a 1.5 cm HealTekPatch™ disk or a2.5 cm square of gauze) was applied to each needle site. Each materialwas held in place, with firm digital pressure, for 5 minutes, afterwhich digital pressure was removed, taking care not to remove the testmaterial. Each site was observed for a period of 2 minutes. If bleedingwas noted within this 2-minute observation period, digital pressure wasimmediately reapplied for an additional 5 minutes. This process wasrepeated until cessation of bleeding was achieved. The number ofapplications of pressure needed for cessation of bleeding was recordedto a maximum of three applications. If bleeding was not stopped within15 minutes of treatment (i.e. three 5-minute pressure applications), thedata was no longer recorded and compression resumed until the bleedingstopped for that patient. The bleed time was defined as the period oftime beginning with removal of the needle from a site and ending whenbleeding from that site had stopped. Arterial and venous bleed timeswere recorded separately and the longer of the two time periods wasconsidered to be the bleed time for the particular treatment. Theresults of this clinical trial are summarized in Table 2, below.

TABLE 2 CLINICAL TRIAL RESULTS TIME (MIN) TO CESSATION PATIENT OFBLEEDING USING: TREATMENT PROVIDING NUMBER PATCH GAUZE IMPROVED RESULTS1 5 10 HealTekPatch ™ 2 5 15 HealTekPatch ™ 3 10   15+ HealTekPatch ™ 45 15 HealTekPatch ™ 5 15   15+ HealTekPatch ™ 6 5  5 Tie* 7 5   15+HealTekPatch ™ 8 10 10 Tie 9 5 10 HealTekPatch ™ 10 5 15 HealTekPatch ™11 15 10 Gauze 12 10   15+ HealTekPatch ™ 13 10 15 HealTekPatch ™ 14 5 5 Tie* 15 5 10 HealTekPatch ™ 16 10 15 HealTekPatch ™ 17 5 10HealTekPatch ™ 18 5  5 Tie* 19 5 10 HealTekPatch ™ 20 5 10HealTekPatch ™ 21 5  5 Tie* 22 5 10 HealTekPatch ™ 23 5 10HealTekPatch ™ 24 5 10 HealTekPatch ™ 25 5 15 HealTekPatch ™ Tie* 4 of 5tie scores occurred in the initial 5 minutes of pressure. Observation ofevents occurring in the initial 5 minutes were beyond the scope of theprotocol.

5.1.4. Summary of Results

As demonstrated in Table 2, above, bleeding times in 19 of 25 patientswere shorter using HealTekPatch in comparison to gauze. With respect tothe remaining six patients, bleeding times were the same using eitherHealTekPatch™ or gauze for 5 patients while the bleeding time observedwas longer for only one patient using HealTekPatch™ as compared to theuse of the control material, gauze.

In addition, it was noted that, surprisingly, it was possible to stopbleeding in less than 15 minutes in the majority of instances usingeither HealTekPatch™ or gauze. This is in contrast to standard procedurefor hemodialysis patients, which comprises applying compression for atleast 15 minutes stop bleeding.

5.1.5. Data Analysis 5.1.5.1. Statistical Evaluation

The data obtained in the clinical trial described above, were analyzedby personnel of the Biostatistics Research Center, New England MedicalCenter (Boston, Mass.), as described below.

For purposes of the initial analysis, each tie was counted as a one-halfsuccess. Accordingly, this analysis was based upon 21.5 successes out of25 trials. This leads to an estimated effectiveness of 86% with an exactbinomial confidence interval of (68%, 96%). A plot of the cumulativeproportion of success for this distribution shows that there is lessthan a 1 in 100 chance that the success rate is less than about 2 in 3.A plot of the posterior distribution of the success proportion wasgenerated using a Bayesian analysis based on a noninformative Beta priordistribution, and showed that the data most likely support a successrate of 86%. This analysis further indicated that a rate less than 70%is unlikely while a rate of success of 95% is certainly possible.

5.1.5.2. Sensitivity Analysis

A second, more conservative analysis was also carried out in which tieswere treated as failures. This analysis provided a success rate of 76%(19 in 25), with an exact confidence interval for this rate of (56%,92%). Even this conservative analysis shows that there is almost nopossibility that use of HealTekPatch™ is not as effective as the gauzecontrol in promoting cessation of bleeding from a vascular access siteafter needle removal.

In a third analysis of the data obtained in the clinical study disclosedabove, four of the ties (i.e., those in which bleeding had been arrestedwithin the first five-minute test period) were eliminated, while thefifth tie was deemed a failure. In this instance, the success rate was93% (19.5 out of 21) with an exact 95% confidence interval of (76%,100%).

5.1.5.3. Wound Closure Using HealTekPatch™

The data of Table 2 demonstrate that application of the HealTekPatch™stopped bleeding within five minutes for 72% (18 of 25) patients, whilethe control, standard application of a gauze square stopped bleedingwithin five minutes only 16% (4 or 25) patients.

5.1.5.4. Statistical Analysis of Wound Closure Using HealTekPatch™

Using the HealTekPatch™ as described in the previous section (5.1.5.3),there were 18 successes out of 25 trials. This leads to an estimatedeffectiveness of 72% with an exact binomial confidence interval of (52%,84%). In contrast, using the standard gauze treatment provided only 4successes out of 25 trials. This leads to an estimated effectiveness of16% with an exact binomial confidence interval of (0%, 28%).

5.2. Example 2 Use of the HealTekPatch™ to Promote Rapid Wound Closureand Cessation of Bleeding in Hemodialysis Patients on ProphylacticAnticoagulant Therapy

Within the set of 25 patients enrolled in the clinical study describedin Example 1, above, there was a subset of 13 patients who were alsobeing treated with prophylactic anticoagulants (aspirin, Ecotrin, orWarfarin). As demonstrated below, rapid wound closure and cessation ofbleeding from vascular access sites was more effectively achieved byapplication of HealTekPatch™ discs than by application of gauze squares.

This conclusion is based upon a review of the bleeding-time dataobtained for the subset of 13 anticoagulant-treated patients of theclinical study of Example 1, above. Accordingly, the materials andtreatment methods of Example 2 are those described in Sections 5.1.1 and5.1.3 of Example 1, above.

5.2.1. Summary of Results

Table 3 summarizes the results obtained in the clinical study describedabove for that subset of patients who were being treated with additionalanticoagulation therapy.

TABLE 3 TIME (MIN) TO CESSATION TREATMENT ADDITIONAL OF BLEEDINGPROVIDING PATIENT ANTICOAGULANT USING: IMPROVED NUMBER ADMINISTEREDPATCH GAUZE RESULTS 1 Aspirin 5 10 HealTekPatch ™ 3 Ecotrin 10   15+HealTekPatch ™ 6 Aspirin 5  5 Tie* 7 Ecotrin 5   15+ HealTekPatch ™ 8Warfarin 10 10 Tie 10 Warfarin 5 15 HealTekPatch ™ 13 Aspirin 10 15HealTekPatch ™ 16 Aspirin 10 15 HealTekPatch ™ 17 Ecotrin 5 10HealTekPatch ™ 18 Aspirin 5  5 Tie* 22 Aspirin 5 10 HealTekPatch ™ 24Warfarin 5 10 HealTekPatch ™ 25 Warfarin 5 15 HealTekPatch ™ (Tie*: Twoof the three tie scores occurred in the initial 5 minutes of pressure).Observation of events occurring in the initial 5-minute time period werebeyond the scope of the protocol followed in this clinical study, asdescribed above in Section 5.1.3.

As indicated in Table 3, bleeding times were shorter when aHealTekPatch™, rather than a gauze square was applied to vascular accesssites after needle removal at the end of a hemodialysis treatmentsession in 10 of 13 patients identified as being treated withprophylactic anticoagulant therapy. Moreover, for the remaining 3patients of this anticoagulant-treated subset of patients, bleedingtimes were the same (i.e. designated as a tie) whether a HealTekPatch™or a gauze square was applied to the vascular access site afterhemodialysis.

5.2.2. Data Analysis

Statistical evaluation of the results of Experiment 2 was performedusing the methods described above in Section 4.5.1, and counting a tieas one-half success. According to this analysis, there were 11.5successes out of 13 trials, which leads to an estimated effectiveness of88% with an exact binomial confidence interval of (62%, 100%).

5.2.3. Conclusion

The clinical study described above in Examples 1 and 2 demonstrates thatHealTekPatch™ is very effective in reducing the duration of postdialysisbleeding from vascular access sites of hemodialysis patients. Treatmentusing HealTekPatch™ proved more effective than treatment with thecontrol material, gauze, in 86% of the patients tested. Use ofHealTekPatch™ material stopped bleeding within 5 minutes in 72% (18 of25) patients, while the standard care (treatment with gauze) stoppedbleeding within 5 minutes in only 16% (4 of 25) patients. Moreover, inthe subset of patients treated with prophylactic anticoagulants,treatment using HealTekPatch™ was more effective than use of the gauzecontrol, for rapid wound closure and cessation of bleeding in 88% of thepatients treated. These results verify that the HealTekPatch™ promotesrapid wound closure and cessation of bleeding in hemodialysis patientsas well as for patients on prophylactic anticoagulation therapy.Accordingly, use of HealTekPatch™ as described above, will facilitateearlier patient ambulation and return to the home environment.

5.3. Example 3 Use of Hemostatic Compositions in Conjunction With theMethods of the Present Invention to Promote Rapid Wound Closure andCessation of Bleeding in Hemodialysis Patient 5.3.1. Introduction

The standard method for achieving hemostasis at vascular access sites ofpatients after hemodialysis treatment involves application of a gauzepad, with pressure, until bleeding stops. In many instances, thispressure must be applied for a period of 15 to 20 minutes, and standardprocedures utilize at least 15-20 minutes of compression. However, asdemonstrated in the data provided below, an extended period ofcompression (i.e. greater than 14 minutes), correlates with vascularaccess complications, which are referred to as “vascular events.” Thatis, patients who require a longer time to control post-hemodialysisbleeding will experience a greater number of vascular accesscomplication events than those who need pressure applied for a shorterperiod of time. Accordingly, use of treatments that reduce the length oftime needed to achieve post-hemodialysis hemostasis, such as thosedescribed in Examples 1 and 2, above, will significantly decrease theincidence of vascular access complications and therefore will extend thelifetime and improve the patency rates of both primary arteriovenousfistulae and synthetic grafts. Reduction in access complications willlead to major cost savings. As noted above, such vascular accesscomplications are the single greatest cause of morbidity within thehemodialysis patient population requiring expensive, extended medicalattention and inpatient care.

5.3.2. Data Collection

The data of Table 4, below, were assembled over an eight-monthobservation period directed toward the 66 patients identified in Example1 above, and were collected from both physical and electronic charts foreach patient. Electronic chart data included that entered in operatingroom notes, special procedure notes, radiology results, and dischargesummaries. Each patient's postdialysis compression time was checkedtwice in one week, every week, every other month for the eight-monthperiod of the study. Postdialysis compression time was defined asextending from removal of the first needle until the cessation ofbleeding at both sites. Concurrent patient data on hematocrit, albumin,platelet count, KTV, PORN, blood flow, and veinous pressure werecollected for each patient to insure that there had been no physicalchange in the patient's health status.

TABLE 4 Patient Compression Months on Number of Number Time (in minutes)Dialysis Vascular Events 24 6.1 37.3 0 2 6.2 37.5 0 65 6.6 20.5 0 63 6.723.4 0 56 6.7 41.0 2 3 6.8 84.5 0 20 8.3 73.2 0 40 8.3 13.6 0 7 9.0 19.60 44 9.2 13.5 0 37 9.3 4.1 0 47 9.3 17.3 1 53 9.5 5.1 0 41 10.0 16.4 0 810.5 29.5 1 50 10.5 18.9 1 6 10.6 36.2 1 17 10.6 35.0 4 25 10.9 28.6 1 911.1 37.3 1 62 11.5 46.9 2 66 11.8 20.9 1 22 12.0 31.0 3 52 12.2 27.6 045 12.3 34.5 0 54 12.3 11.2 0 27 12.7 38.7 0 16 13.3 9.2 0 10 14.0 12.51 Total: 812.5 Total: 15 58 14.1 24.4 1 34 14.2 16.7 0 35 15.2 8.3 0 4915.2 35.5 0 61 15.2 13.4 2 31 15.5 49.2 1 55 15.8 23.1 2 38 15.8 49.2 342 16.0 38.3 0 36 16.1 29.7 3 15 16.2 27.5 0 46 16.2 17.4 0 57 16.3 42.51 39 16.7 16.4 0 51 16.7 56.0 4 12 17.1 51.4 1 59 17.7 64.5 3 4 18.341.6 0 28 18.3 33.5 1 33 18.5 40.5 1 43 18.8 31.6 2 26 19.1 31.1 2 520.5 18.4 0 14 20.5 39.1 1 18 20.6 22.8 2 21 20.8 27.0 1 29 21.0 15.7 260 22.2 36.3 1 1 22.3 47.0 2 30 22.5 14.8 0 13 22.6 11.2 1 32 22.8 32.51 19 24.1 18.8 0 64 26.0 8.4 1 23 27.1 18.5 0 18 34.3 31.5 0 11 48.033.4 1 Total: 1129.7 Total: 38

5.3.3. Summary of the Data Correlating Compression Time and Frequency ofVascular Events

The average compression time required to control postdialysis bleedingwas observed to be 15.5 minutes. Surprisingly, 69.5% of the vascularaccess events, i.e., vascular access complications, occurred in thosepatients requiring compression for more than 14 minutes. Morespecifically, the data of Table 4 indicate that a total of 18 vascularevents were noted within a total of 812.5 months of medical care of 29patients requiring less than 14 minutes of standard treatment to achievehemostasis subsequent to a dialysis session. In contrast, the data ofTable 4 indicate that a total of 41 vascular events were noted within atotal of 1129.7 months of medical care of the remaining 37 patients inthe study who required 14 minutes or longer of standard treatment toachieve hemostasis subsequent to a dialysis session. The 63.6% increase[(41/1129.7)/(18/812.5)] in the incident rate of vascular eventoccurrences per month for those patients requiring at least 14 minutesof compression is striking.

Accordingly, treatment methods, such as those described in Examples 1and 2 above, that would decrease the length of time of compressionrequired to achieve hemostatis within the patient population requiring14 minutes or more of compression to achieve hemostatis, would decreasethe number of vascular events observed over the long term. In addition,the treatment methods, such as those described in Examples 1 and 2above, that would decrease the length of time of compression required toachieve hemostatis within the patient population requiring 10 minutes ormore of compression to achieve hemostatis, would also decrease thenumber of vascular events observed over the long term.

5.3.4. Discussion

The results of the experiments presented hereinabove demonstrate, first,that the length of compression time at the site of hemodialysis needlepunctures is the cause of vascular access complications, and second,that the length of compression time needed to cease or reduce bleedingcan be reduced by application of the methods of the invention.

One of ordinary skill in the art would not have expected a reducedcompression time at the site of removal of hemodialysis needles forabout one to fourteen minutes could reduce vascular access complicationsat the site where the vein and artery have been sutured or where thesynthetic graft is connected to the blood vessels.

It is counterintuitive to expect a direct correlation between vascularaccess complications and the extent, i.e. amount or length, ofcompression. This is particularly true for vascular access complicationsat the site where the artery and vein have been connected in an AVfistulae or the site of attachment of graft. The primary cause of accessfailure is thrombosis and longer compression is indicative ofcoagulopathy. Furthermore, the generally accepted pathology associatedwith vascular access failure has been found to be intimal hyperplasia atthe venous anastamosis site not at the cannulation site. Thus, one ofordinary skill in the art of hemodialysis would not expect to obtain adirect and quantitative relationship between the length of time pressureis applied to a vascular access site to control bleeding and the numberof vascular access complications in hemodialysis patients.

While applying excessive or prolonged compression to the site where thehemodialysis needles have been removed is known to cause thrombosis andother vascular complications at the site where compression is applied itis surprising that reducing compression to about one to fourteenminutes, for the majority of hemodialysis sessions could reduce vascularaccess complications at the site where, for example, the A-V fistulaehas been created or where the synthetic graft is connected to the bloodvessels.

5.4. Example 4 Use of the HealTekPatch™ to Promote Rapid Wound Closureand Cessation of Bleeding in Eighty Hemodialysis Patients

Example 4 is also directed toward a study testing the utility of aparticular material, HealTekPatch™, as a device to promote rapid woundclosure and cessation of bleeding from such vascular access sites inhemodialysis patients following needle removal.

The study presented in Example 4 differs from that of Example 1 inseveral aspects, including, but not limited to, the number ofhemodialysis patients included in the study, and the method for timingcessation of bleeding. As indicated by the data provided below,HealTekPatch™ resulted in a 68% reduction in the average time requiredachieve cessation of bleeding.

5.4.1. Materials and Methods

HealTekPatch™, (Marine Polymer Technologies, Danvers, Mass.) afully-acetylated poly-N-acetyl glucosamine (p-GlcNAc), is a highlypurified polysaccharide free of protein and other contaminants, seeSections 4.1.4 and 5.1.1

5.4.1.1. Patient Selection

Eighty patients from four hemodialysis centers were monitored to assessthe time required to obtain cessation of bleeding in hemodialysispatients and estimate reductions in vascular access complications.Patients were offered the opportunity to participate in the study whenthey presented for routine hemodialysis. The patients monitored met thefollowing inclusion criteria: male and female age of 18 years and above,having chronic renal failure, undergoing chronic hemodialysis,functional vascular access as confirmed by Doppler examination, andhaving signed an Informed Consent Form. Patient evaluations wereconducted the same as described for Example 1 to ensure that patientsmet study inclusion criteria and to collect relevant clinical data. (SeeSection 5.1.2).

5.4.2. Treatment Method

Each patient served as his/her own control receiving either a hemostaticpatch or standard of care gauze control to achieve hemostasis. The orderin which each material (HealTekPatch™ or gauze square) was used, wasrandomized for each patient. Following the removal of the dialysisneedles, the randomly selected treatment (either a HealTekPatch™ orgauze control) was applied to each needle site. These were held in placewith firm digital pressure for 5 minutes. Then the digital pressure wasremoved, taking care not to remove the test material. The sites wereobserved for a period of 2 minutes. If bleeding recurred during the 2minute period of observation, digital pressure was reapplied immediatelyfor an additional 1 minute. This was repeated until cessation ofbleeding was achieved. If the patient reached 30 minutes withouthemostasis, the treatment was interrupted and assigned a value of 30.The time required for cessation of bleeding was recorded. The bleed timewas considered the time from the removal of the needle until the sitestopped bleeding. Arterial and venous bleed times were recordedseparately and the longer of the two lengths of time was considered tobe the bleed time for the particular treatment. Vascular AccessComplications were estimated by applying the differential in timerequired to achieve hemostasis to the regression analysis described inExample 1.

5.4.3. Results

Results from the trial are presented in Table 5. The use of theHealTekPatch™ resulted in a 68% reduction in the average time requiredachieve cessation of bleeding. The time differences were statisticallysignificant.

TABLE 5 TIME (MIN) TO CESSATION OF BLEEDING USING TIME DIFFERENCEPATIENT AGE GAUZE PATCH MINUTES % 1 31 15 5 10 66.7% 2 61 18 6 12 66.7%3 53 20 6 14 70.0% 4 89 22 5 17 77.3% 5 74 25 6 19 76.0% 6 53 15 5 1066.7% 7 50 18 5 13 72.2% 8 55 18 5 13 72.2% 9 45 15 5 10 66.7% 10 73 195 14 73.7% 11 84 19 5 14 73.7% 12 68 15 5 10 66.7% 13 51 15 5 10 66.7%14 60 16 5 11 68.8% 15 61 15 5 10 66.7% 16 59 22 5 17 77.3% 17 77 18 513 72.2% 18 74 20 7 13 65.0% 19 71 15 5 10 66.7% 20 73 16 5 11 68.8% 2132 15 5 10 66.7% 22 29 15 5 10 66.7% 23 53 16 5 11 68.8% 24 70 18 5 1372.2% 25 59 20 5 15 75.0% 26 53 19 5 14 73.7% 27 83 18 5 13 72.2% 28 6215 5 10 66.7% 29 48 16 5 11 68.8% 30 69 16 5 11 68.8% 31 57 19 7 1263.2% 32 79 17 6 11 64.7% 33 61 16 7 9 56.3% 34 69 23 7 16 69.6% 35 6829 7 22 75.9% 36 18 19 5 14 73.7% 37 54 15 6 9 60.0% 38 67 25 7 18 72.0%39 39 23 8 15 65.2% 40 83 25 8 17 68.0% 41 55 30 8 22 73.3% 42 72 26 818 69.2% 43 50 22 8 14 63.6% 44 47 27 8 19 70.4% 45 57 23 7 16 69.6% 4652 20 7 13 65.0% 47 70 16 7 9 56.3% 48 51 22 7 15 68.2% 49 72 16 5 1168.8% 50 62 15 6 9 60.0% 51 76 20 7 13 65.0% 52 60 15 6 9 60.0% 53 66 207 13 65.0% 54 69 23 7 16 69.6% 55 33 24 8 16 66.7% 56 64 24 8 16 66.7%57 72 23 7 16 69.6% 58 36 16 6 10 62.5% 59 66 23 6 17 73.9% 60 67 15 7 853.3% 61 69 18 6 12 66.7% 62 79 13 7 6 46.2% 63 74 19 7 12 63.2% 64 6223 7 16 69.6% 65 52 23 8 15 65.2% 66 76 24 8 16 66.7% 67 64 30 6 2480.0% 68 70 25 8 17 68.0% 69 66 16 5 11 68.8% 70 67 30 8 22 73.3% 71 5516 7 9 56.3% 72 77 20 6 14 70.0% 73 70 30 7 23 76.7% 74 73 11 6 5 45.5%75 84 10 5 5 50.0% 76 59 10 6 4 40.0% 77 58 10 5 5 50.0% 78 42 14 6 857.1% 79 66 10 5 5 50.0% 80 60 11 6 5 45.5% average 19 6 13 68.4%

All 80 patients exhibited a reduction in time to cessation of bleedingusing the HealTekPatch™ in comparison to the control. The data in Table5 also demonstrate that that application of the HealTekPatch™ stoppedbleeding within five minutes for 40% (32 of 80) of patients, within 6minutes for 60% (48 of 80) patients, and within 7 minutes for 85% (68 of80) of patients.

5.4.4. Correlating Compression Time and Frequency of Vascular Events

To examine the relationship between compression time and vascular accesscomplications, a Poisson regression model was constructed to relate thenumber of vascular access complications to the compression time requiredto achieve hemostasis. In this analysis the logarithm of the number ofvascular access complication events per month was related to thecompression time required for Blacks and non-Blacks separately by thefollowing two equations:

log(events/month)=−7.042+0.367*compression time(non-Blacks)

log(events/month)=−6.737+0.367*compression time(Blacks)

The distribution of reductions in compression times were as follows: 29patients of 80 (36%) exhibited reduced compression times from 11 minutesor greater to 5 minutes; 3 patients of 80 (3%) exhibited reducedcompression times from 10 minutes to 5 minutes; 15 patients of 80 (18%)exhibited reduced compression times from 11 minutes or greater to 6minutes; 1 patients of 80 (1%) exhibited reduced compression times from10 minutes to 6 minutes; 20 patients of 80 (25%) exhibited reducedcompression times from 11 minutes or greater to 7 minutes; and 12patients of 80 (15%) exhibited reduced compression times from 11 minutesor greater to 8 minutes. Using the regression equations above, each ofthese 80 patients would be expected to have a proportionate reduction innumbers of vascular access complication events. The benefit for eachpatient was calculated as follows:

exp(0.367*#minutes reduced time less than 11 minutes).

Weighting by the distributions of time reductions gives the followinggeometric mean benefit:

exp{0.36*(0.367*6)+0.03*(0.367*5)+0.18*(0.367*5)+0.01*(0.367*4)+0.25*(0.367*4)+0.15*(0.367*3)}=5.61

Thus on average, the number of access events is estimated to be reducedby a factor of 5.61.

5.4.5. Conclusion

The clinical study described above in Example 4 coberates the results ofExamples 1 and 2 in demonstrating that HealTekPatch™ is very effectivein reducing the duration of postdialysis bleeding from vascular accesssites of hemodialysis patients. Treatment using HealTekPatch™ provedmore effective than treatment with the control material, gauze, in allof the patients tested. Use of HealTekPatch™ material resulted in a 68%reduction in the average time required achieve cessation of bleeding

Use of HealTekPatch™ material also stopped bleeding in an average of 6minutes, while the standard care (treatment with gauze) stopped bleedingin an average of 19 minutes. Moreover, the reduction in compression timewas estimated to be proportionate to the reduction in numbers ofvascular access complication events. Accordingly, use of HealTekPatch™as described above, will facilitate earlier patient ambulation,reduction in vascular access complications, and increased viability andlongevity of vascular access sites.

5.5. Example 5 Regression Analysis in Hemodialysis Patients

Age, gender, race, vascular access, type of anticoagulant, duration ofdialysis, diabetes and hypertension were evaluated as potentialcovariate factors with length of compression time in a multipleregression model.

5.5.1. Materials and Methods 5.5.1.1. Patient Selection

Sixty-six hemodialysis patients were monitored to assess the timerequired to obtain cessation of bleeding in hemodialysis patients andestimate reductions in vascular access complications. Patients wereoffered the opportunity to participate in the study when they presentedfor routine hemodialysis. The patients monitored met the followinginclusion criteria: male and female age of 18 years and above, havingchronic renal failure, undergoing chronic hemodialysis, functionalvascular access as confirmed by Doppler examination, and having signedan Informed Consent Form. Patient evaluations were conducted the same asdescribed for Example 1 to ensure that patients met study inclusioncriteria and to collect relevant clinical data. (See Section 5.1.2).Compression time was measured by the same methods as described insection 5.4.2 and average compression times were calculated for eachpatient and the patient population.

5.5.1.2. Preliminary Analyses

A preliminary screening analysis was conducted to determine therelationship between each of the above-mentioned factors and the numberof vascular access events more than four months after dialysis forpatients with average compression times less than 30 minutes. Three ofthe 66 patients are excluded: two had compression times greater than 30minutes and one was followed for fewer than four months after dialysis.An unadjusted p-value was determined using a nonparametric test: theWilcoxon rank-sum test for the above-mentioned factors with two levelsand the Kruskal-Wallis test for factors with more than two levels.Adjusted p-values for the above-mentioned factors were based on aPoisson regression model adjusting for time on dialysis.

A second preliminary screening analysis was conducted to determine theSpearman rank correlations between continuous measured variables and thenumber of vascular access events more than four months after dialysisfor patients with average compression times less than 30 minutes. Anunadjusted p-value for the measured variables was derived from theSpearman rank correlation. (Reference: Miller R G. Beyond ANOVA, Basicsof Applied Statistics. Wiley, New York, 1986). In both tables, theadjusted p-value refers to the factor tested in a Poisson regressionadjusted for the time on dialysis. Continuous variables were includedusing a smoothing spline representation that permits arbitrary nonlinearshapes. (Reference: Hastie T J and Tibshirani R J. Generalized AdditiveModels Chapman and Hall, N.Y., 1990). Adjusted p-values were based on aPoisson smoothing spline model adjusting for time on dialysis. Thismodel is described further below.

5.5.1.3. Multiple Regression Analysis

A multiple regression analysis was performed to describe the variationin the number of events that occur more than four months after the startof dialysis in terms of a Poisson model. The Poisson model assumes thatevents within groups of patients defined by the regression variablesoccur independently at random times over the course of the study. ThePoisson models were fit as generalized linear models with a log link(Reference: McCullagh P and Nelder J A. Generalized Linear Models, 2nded. Chapman and Hall, N.Y., 1989) using the S-Plus software (StatSci,data analysis products division of MathSoft, Inc., Seattle, Wash.). Theresponse was the number of events occurring more than four months afterthe start of dialysis and the number of months on dialysis beyond theinitial four was used as an offset in the Poisson model formulation toadjust for time on dialysis. This offset converts the outcome to anevent rate so that the log event rate=log(events/time) is a regressionfunction of the compression time and other covariates. The expectedevent rate was then expressed as a sum of components due to thesecovariates. Since patients with longer dialysis histories were morelikely to have vascular access complication events, all regressionanalyses were expressed in terms of event rates, the number of eventsper month.

The best regression function was obtained by examining each of thevariables listed in Tables 1 and 2 in candidate models. Discrete factorswere fit with standard dummy variable techniques. Continuous variableswere examined as smoothing splines using generalized additive modelmethods to assess potential nonlinearities. Appropriate parametric formsof these factors were then chosen if deemed significant.

In a Poisson regression, the deviance (or −2*log likelihood) is ameasure of the goodness of fit of the model. It is expected to be closeto the number of degrees of freedom (the sample size N minus the numberof parameters p) in a well-fit model (Reference: McCullagh P and NelderJA. Generalized Linear Models, 2nd ed. Chapman and Hall, N.Y., 1989).

For given race and compression time, the model then gives that vascularaccess events follow a Poisson distribution with an expected event rate.The logarithm of the number of vascular access complication events permonth was related to the compression time required for Blacks andnon-Blacks separately by the two equations described above in Section5.4.4.

The predicted event rates are then calculated by taking theantilogarithm of the log event rates. Standard errors for these logpredicted values may be calculated as:

standard error=√[Var(a)+Var(b)*T ²+2*T*Cov(a,b)]

where a is the intercept, b is the slope, T is the compression time, Varindicates the variance and Cov indicates the covariance. The upper andlower limits of the 95% confidence interval are then calculated from theantilogarithm of the estimate±1.96*standard error.

5.5.2. Results

The factors for each patient, compression time, duration of dialysis inmonths, and number of vascular access complications are presented inTable 6. These data were then used in the preliminary analyses andmultiple regression analysis.

TABLE 6 Diabetes Compres- Months No. of Patient Hyper- Anticoag Fistulasion On Procedures Events/ No. Sex Age Race Tension Therapy or GraftTime Dialysis (Events) Mos Dial 24 M 58 B D N F 6.1 37.3 0 0.000 2 F 72B N F 6.2 37.5 0 0.000 65 M 26 W N F 6.6 20.5 0 0.000 63 M 68 W E F 6.723.4 0 0.000 56 M 35 W N F 6.7 41.0 2 0.049 3 F 81 B H N G 6.8 84.5 00.000 20 M 46 W A F 8.3 73.2 0 0.000 40 M 77 A H N F 8.3 13.6 0 0.000 7F 66 H D/H W F 9.0 19.6 0 0.000 44 M 58 W N F 9.2 13.5 0 0.000 37 F 68 WN F 9.3 4.1 0 0.000 47 F 56 B D A F 9.3 17.3 1 0.058 53 M 63 B D A F 9.55.1 0 0.000 41 M 83 A D E F 10.0 16.4 0 0.000 8 M 65 A H N F 10.5 29.5 10.034 50 M 71 B N F 10.5 18.9 1 0.053 6 M 75 W E F 10.6 36.2 1 0.028 17M 65 W N G 10.6 35.0 4 0.114 25 M 38 B N F 10.9 28.6 1 0.035 9 M 85 WH/D N F 11.1 37.3 1 0.027 62 F 69 W N G 11.5 46.9 2 0.043 66 M 68 A D WF 11.8 20.9 1 0.048 22 F 37 B N F 12.0 31 3 0.097 52 M 65 W H A F 12.227.6 0 0.000 45 M 56 W D N F 12.3 34.5 0 0.000 54 M 41 B N F 12.3 11.2 00.000 27 F 49 B D N F 12.7 38.7 0 0.000 16 M 76 W D W F 13.3 9.2 0 0.00010 M 84 A N F 14.0 12.5 1 0.080 58 F 81 B H N G 14.1 24.4 1 0.041 34 F63 B H E G 14.2 16.7 0 0.000 35 M 86 W A F 15.2 8.3 0 0.000 49 M 87 A HE F 15.2 35.5 0 0.000 61 F 65 B D E G 15.2 13.4 2 0.149 31 F 48 A N F15.5 49.2 1 0.020 55 M 78 W N F 15.8 23.1 2 0.087 38 M 60 W N G 15.849.2 3 0.061 42 F 77 A H A F 16.0 38.3 0 0.000 36 F 40 B N G 16.1 29.7 30.101 15 F 78 W A G 16.2 27.5 0 0.000 46 F 31 H N G 16.2 17.4 0 0.000 57F 84 B H N G 16.3 42.5 1 0.024 39 F 76 A H/D E F 16.7 16.4 0 0.000 51 F86 A N F 16.7 56.0 4 0.071 12 F 80 A N F 17.1 51.4 1 0.019 59 M 55 W D WG 17.7 64.5 3 0.046 4 F 69 W W G 18.3 41.6 0 0.000 28 F 84 A N F 18.333.5 1 0.030 33 F 65 H D N F 18.5 40.5 1 0.025 43 M 40 W D N F 18.8 31.62 0.063 26 M 68 W H W G 19.1 31.1 2 0.064 5 M 63 B H A F 20.5 18.4 00.000 14 M 49 W D N F 20.5 39.1 1 0.026 18 M 65 B D N G 20.6 22.8 20.088 21 F 69 W E G 20.8 27.0 1 0.037 29 M 73 A W G 21.0 15.7 2 0.128 60M 48 A H W F 22.2 36.3 1 0.028 1 M 62 W W G 22.3 47.0 2 0.043 30 M 64 BD N F 22.5 14.8 0 0.000 13 F 70 W D E G 22.6 11.2 1 0.090 32 F 62 B D WG 22.8 32.5 1 0.031 19 F 69 W D A F 24.1 18.8 0 0.000 64 F 85 B W G 26.08.4 1 0.119 23 F 76 A W F 27.1 18.5 0 0.000 48 M 73 W D N F 34.3 31.5 00.000 11 M 78 A D N F 48.0 33.4 1 0.030 Legend M Male F Female B Black WWhite H Hispanic A Asian A Aspirin E Ecotrin W Warfarin N None

The results of the preliminary analyses presented in Tables 7 and 8.Table 7 shows the effect of factors on number of vascular access eventsmore than four months after dialysis for patients with averagecompression times less than 30 minutes. Table 8 shows the Spearman rankcorrelations between continuous measured variables and the number ofvascular access events more than four months after dialysis for patientswith average compression times less than 30 minutes. The resultstogether show that only race and compression time are significant bothbefore and after adjusting for duration of dialysis, which was a highlysignificant factor. Black patients and patients with longer compressiontimes were more likely to experience vascular access complications. Theadjustment for duration of dialysis had little effect on the othercovariates.

TABLE 7 Number of Events P-value N 0 1 2 3 4 Unadjusted Adjusted OVERALL63 27 21 9 4 2 By Gender 0.83 0.81 Female 28 11 10 6 1 0 Male 35 16 11 33 2 By Site 0.42 0.42 Fistula 42 19 15 4 2 2 Graft 21 8 6 5 2 0 ByAnticoagulant 0.28 0.37 A 8 3 4 1 0 0 C 11 7 2 1 1 0 N 35 12 12 6 3 2 E9 5 3 1 0 0 By Race 0.31 0.17 Asian 15 6 6 1 2 0 Black 18 6 4 6 1 1Hispanic 3 2 1 0 0 0 White 27 13 10 2 1 1 By Black Race 0.11 0.04 ByDiabetes 0.32 0.84 Yes 22 12 5 3 1 1 No 41 15 16 6 3 1 By Hypertension0.69 0.18 Yes 15 6 7 2 0 0 No 48 21 14 7 4 2

TABLE 8 P-value Variable Correlation Unadjusted Adjusted Age −0.07 0.580.36 Compression Time 0.23 0.07 0.10 Duration of Dialysis 0.34 0.007

The results of the multiple Poisson regression model analysis forvascular access events more than four months after dialysis for patientswith average with compression times of less than 30 minutes arepresented in Table 9. The results show that only the covariates ofcompression time and race (Black/not Black) were significant predictorsin a multiple regression model. Using a generalized additive model, asmoothing spline representation for compression time was obtained whichwas then approximated by a piecewise linear function such that longercompression times increased the event rate up to 11 minutes and then hadno further effect. The inclusion of this piecewise function and thefactor for race reduced the deviance to 60.7 on 60 degrees of freedomindicating that the remaining lack of fit could be taken as randomPoisson variation. In a null model without any covariates, the deviancewas 71.5 on 62 degrees of freedom showing that the counts were nearlyPoisson (i.e., the events were nearly random) without any adjustment orstratification.

TABLE 9 Variable Coefficient Standard Error P-value Intercept −7.0421.837 Compression Time* 0.367 0.170 0.03 Black Race 0.305 0.133 0.02*Compression Time is truncated at 11 minutes. Null Deviance: 71.47239 on62 degrees of freedom Residual Deviance: 60.66224 on 60 degrees offreedom Correlation of Regression Coefficients Intercept withcompression time: −0.997 Intercept with race: −0.019 Compression timewith race: 0.035

The results of the logarithmic relation of the number of vascular accesscomplication events per month to the compression time required forBlacks and non-Blacks separately indicated that, adjusting for time ondialysis, an increase of one minute in the compression time (up to 11minutes) increased the log number of events by 0.367 (standarderror=0170) or the number of events by a factor of 44% (95% CI=3%-101%)for all patients. There was an increase of 36% (95% CI=5%-76%) for Blackpatients compared with non-Black patients at all compression times.There was no significant interaction between these two factors.

The results of the analyses presented herein indicate that from apractical and clinical standpoint compression times of 5 minutes of lessare optimal, between 5 and 10 minutes resulted in incremental damage tothe vascular access site and compressions greater than 10 minutes weredamaging to the vascular access site. The relationship betweencompression time and vascular access complications determined usinggauze alone can serve as a baseline from which to compare the methods ofthe invention which use a vasoconstrictor or coagulant rather than gauzeor a bandage without a vasoconstrictor or coagulant.

5.6. Example 6 Use of the HealTekPatch™ to Reduce Morbidity inHemodialysis Patients

Example 6 is also directed toward a study that measures morbidity whenutilizing the HealTekPatch™ as a device to promote rapid wound closureand cessation of bleeding from such vascular access sites inhemodialysis patients following needle removal.

The study presented in Example 6 differs from that of Example 4 inseveral aspects, including, but not limited to, the number ofhemodialysis patients in the study, the frequency of recording ofcompression time data, and the recording of morbidity.

5.6.1. Materials and Methods

HealTekPatch™ (Marine Polymer Technologies, Danvers, Mass.), afully-acetylated poly-N-acetyl glucosamine (p-GlcNAc), is a highlypurified polysaccharide free of protein and other contaminants (seeSections 4.1.4 and 5.1.1).

5.6.1.1. Patient Selection

Three hundred hemodialysis patients are monitored for about 30 months toone year to assess the time required to obtain cessation of bleeding inhemodialysis patients and estimate reductions in vascular accesscomplications. Patients are offered the opportunity to participate inthe study when they are presented for routine hemodialysis. The patientsmonitored meet the following inclusion criteria: male and female age of18 years and above, having chronic renal failure, undergoing chronichemodialysis, functional vascular access as confirmed by Dopplerexamination, and having signed an Informed Consent Form. Patientevaluations are conducted the same as described for Example 1 to ensurethat patients meet study inclusion criteria and to collect relevantclinical data. (See Section 5.1.2). Compression time is measured by thesame methods as described in section 5.4.2 and average compression timesare calculated for each patient and the patient population.

5.6.2. Treatment Method

The treatment method is practiced the same as described in Example 4with the exception that actual measurements of compression time toachieve hemostasis are not recorded for all 300 hemodialysis patientsfor each hemodialysis session (see Section 5.4.2). Instead, for eachpatient, compression times are recorded at six week intervals. Death isrecorded and average morbidity was calculated as a percentage of thepatient population.

5.6.3. Results

For a one year study of hemodialysis patients, the expected percentmorbidity is 20% where gauze alone without a coagulant orvasoconstrictor is applied, resulting in longer compression times toachieve hemostasis. For hemodialysis patients where gauze alone is usedwithout a coagulant or vasoconstrictor is applied, resulting in longercompression times to achieve hemostasis, the 5-year survival rate is 29%of patients. The expected percent morbidity is below 20%, and theexpected 5-year survival rate of patients is 30% or greater, when themethods of the invention are practiced for one year or longer.

It is apparent that many modifications and variations of this inventionas set forth here may be made without departing from the spirit andscope thereof. The specific embodiments described above are given by wayof example only, and the invention is limited only by the terms of theappended claims.

Various publications are cite herein, the disclosures of which areincorporated by reference in their entireties.

1.-64. (canceled)
 65. A method for reducing complications at a vascularaccess site associated with hemodialysis therapy in a human patient atrisk for said complications, said method comprising: (a) applyingtopically to the skin at a site where hemodialysis needles have beenremoved in said patient during a hemodialysis session a barrier-formingmaterial comprising poly-β-1→4-N-acetylglucosamine; and (b) applyingcompression to or proximal to the site where the hemodialysis needleshave been removed for a duration of about one to about fourteen minutes.66. The method of claim 65, wherein the complication is a long termvascular access complication.
 67. The method of claim 66, wherein thelong term vascular access complication is hyperplasia, thrombosis,venous stenosis, arterial stenosis, morbidity, or a reduction in bloodflow through the vascular access site relative to a newly maturedvascular access site.
 68. The method of claim 65, wherein thecomplication is a short term vascular access complication.
 69. Themethod of claim 68, wherein the short term vascular access complicationis hematoma or infection.
 70. The method of claim 65, wherein thereduction in complications is a reduction in infection rate.
 71. Themethod of claim 70, wherein the infection is an HIV, a Hepatitis C, avancomycin resistant enterococcus, ormacrolide-lincosamide-streptogramin B (“MLS”)-resistant bacterialinfection.
 72. A method for reducing the failure rate of a vascularaccess site associated with hemodialysis therapy in a human patient,said method comprising: (a) applying topically to the skin at a sitewhere hemodialysis needles have been removed in said patient during ahemodialysis session a barrier-forming material comprisingpoly-β-1→4-N-acetylglucosamine; and (b) applying compression to orproximal to the site where the hemodialysis needles have been removedfor a duration of about one to about fourteen minutes.
 73. A method forreducing the average number of interventions to repair or replace avascular access site associated with hemodialysis therapy in a humanpatient, said method comprising: (a) applying topically to the skin at asite where hemodialysis needles have been removed in said patient duringa hemodialysis session a barrier-forming material comprisingpoly-β-1→4-N-acetylglucosamine; and (b) applying compression to orproximal to the site where the hemodialysis needles have been removedfor a duration of about one to about fourteen minutes.
 74. The method ofclaim 65, 72 or 73, further comprising repeating steps a) and b) for themajority of multiple hemodialysis therapy sessions.
 75. The method ofclaim 65, 72 or 73, wherein the vascular access site is a vein suturedto an artery, a native arteriovenous fistula, or a synthetic vasculargraft.
 76. The method of claim 65, 72 or 73, wherein the compression ismaintained for about one to about ten minutes.
 77. The method of claim65, 72 or 73, wherein the hemodialysis therapy sessions are administeredfor at least three months with 1 or more hemodialysis sessions a week.78. The method of claim 65, 72 or 73, further comprising repeating stepsa) and b) for at least 10 times.
 79. The method of claim 65, 72 or 73,wherein the barrier-forming material is formulated as a mat, a bandage,a patch, or a gauze.
 80. The method of claim 65, 72 or 73, wherein thepoly-β-1→4-N-acetylglucosamine comprises at least 40% ofN-acetylglucosamine monosaccharides that are acetylated.
 81. The methodof claim 65, 72 or 73, wherein the poly-β-1→4-N-acetylglucosamine isfully-acetylated.
 82. The method of claim 65, 72 or 73, wherein thepoly-β-1→4-N-acetylglucosamine is derived from microalgae.
 83. Themethod of claim 65, 72 or 73, wherein the poly-β-1→4-N-acetylglucosamineis biocompatible.
 84. The method of claim 65, 72 or 73, wherein thepoly-β-1→4-N-acetylglucosamine is free of protein.